Against Asiatic Corn Borer (Ostrinia Furnacalis)

EFFICACY OF INSECT-PROTECTED MAIZE (BT-11) AGAINST ASIATIC CORN BORER (OSTRINIA FURNACALIS)

EUFEMIO T RASCO JR1, JHOANAVI MANGUBAT1, ARISTOTLE BURGONIO2, MANUEL LOGRONO2, VIOLETA VILLEGAS2 & EDUARDO °C FERNANDEZ2 1 University of the Philippines in Mindanao, 2 Syngenta Philippines Inc. [email protected]

Three experimental hybrid versions of NK5445 Bt, a hybrid maize developed by Syngenta Seed Company, along with 3 checks were subjected to repeated artificial infestation with Asiatic corn borer (ACB) egg masses and neonate larvae in a screenhouse study to test the efficacy of Bt-11 containing the truncated gene cry1Ab. Results show that all the 3 experimental hybrids were consistently highly resistant to ACB across 3 assessment periods during the vegetative stage. In contrast, the 3 check entries (non-Bt isogenic hybrid of NK5445, sweet corn hybrid Sugar 73, and NK5468, a commercial non-Bt hybrid), showed several elongated feeding holes on as much as two-thirds of the leaves. At harvest, the checks showed severe damage to the stalks, ears, ear shanks and tassels, and relatively minor damage to the kernel. Pupae of ACB were observed inside the stalks. The NK5445 Bt entries showed little or no damage to the stalk, ear shanks, ears, tassels, and kernels. The data clearly demonstrated the efficacy of Bt-11 in controlling the ACB under screenhouse conditions. The presence of 4 other insect species in both checks and NK5445 Bt plants, possibly introduced through the field-collected egg masses used for infestation, is an indication of the safety of the use of Bt-11 concerning non-target insects.

Asiatic corn borer, Bt-11, Bt maize, cry1Ab, genetically modified organism, NK5445, Ostrinia furnacalis, transgenic crop

INTRODUCTION increase due to the use of Bt maize. With a 60% reduction in pesticide use, Bt maize gave an Insect-protected maize hybrids (Bt maize) are incremental profit of P10,132 per hectare. among the products of modern biotechnology that Monsanto is not the only maize seed company have proven to be effective in controlling insect pests that is developing Bt maize technology. Syngenta’s Bt and consequently, reducing pesticide usage, increasing maize, using a similar gene (cry1Ab) that Monsanto yield, and reducing mycotoxin contamination of corn uses in its hybrid, is now increasingly being used by kernels (Phipps & Park 2002, Fernandez et al 1997). maize farmers to control European corn borer in the In 2004, Bt maize was planted in 11.2 million hectares USA and Canada. Syngenta aims to introduce its own worldwide, representing 14% of hectarage of all Bt maize in the Philippines. Towards this objective, transgenic crops (James 2005). Syngenta signed a Memorandum of Agreement with The Department of Agriculture of the Philippines the University of the Philippines in Mindanao to test recognized the importance of Bt maize when it gave the efficacy of its Bt maize (Bt-11) against the ACB. approval for commercial planting of Monsanto’s Bt Considering available information about Bt-11, maize in December 2002. Aside from other data, the including the results of various tests that it has been basis of this approval was the demonstrated efficacy of previously subjected to (Essential Biosafety 2002), a this material against the Asiatic corn borer (ACB) screenhouse study on the Syngenta Bt maize was under Philippine conditions. In a study covering conducted by UP Mindanao in Bago-Oshiro, Davao 10,000 hectares of Bt maize in the Philippines during City, Philippines during the period 15 December 2002 the years 2003 and 2004, Yorobe reported a 37% yield to 28 March 2003. The objective of this study was to test the efficacy of Bt-11 maize containing the Artificial infestation truncated gene cry1Ab against the ACB under The experimental plants were artificially infested screenhouse conditions, the results of which would with egg masses of ACB at 27 DAP. This was repeated become the basis for recommendation of further steps at 41 DAP. One egg mass attached to a 1 x 1 cm leaf to take. piece was dropped on the whorl of the corn plant on the first day. Additional egg masses were applied on MATERIALS & METHODS the second and third days; thus a total of 3 egg masses (approximately 100 eggs) were applied per plant Test materials during each infestation during the vegetative stage. Three experimental hybrid versions of Bt-11 were The egg masses were collected from cornfields in used in this study. These were NK5445Bt (A), General Santos City 1 to 2 days before infestation. NK5445Bt (B), and NK5445Bt (C). For comparison, the When they reached blackhead stage, they were kept in following checks were used: NK5445 non-Bt (isogenic a refrigerator until they were used. hybrid of NK5445), sweet corn hybrid Sugar 73, and Starting at early reproductive stage (55 DAP) to 69 NK 5468. Seeds of the 6 entries were planted in 10 clay DAP, tassels and silks were artificially infested with

Table 1. Guthrie et al (1960) feeding damage scale

Score Resistance Description Rating 1 HR No visible injury or a small amount of pin or fine shot-hole type of injury on a few leaves 2 Small amount of shot-hole type lesions on a few leaves 3 R Shot-hole injury common on several leaves 4 Several leaves with shot-hole and elongated lesions 5 I Several leaves with elongated lesions 6 S Several leaves with elongated lesions about 2.5 cm 7 Long lesions common on about one half of the leaves 8 HS Long lesions common on about 2/3 of the leaves 9 Most leaves with long lesions pots (12-inch diameter) per entry, each pot neonate larvae (total of 15 neonate larvae per silk and representing a replicate in a completely randomized tassel) using camel hairbrush. Most of the tassels were experimental design. Initially, 4 seeds were planted infested at 55 DAP, while silk infestation was more per pot; the plants were subsequently thinned to 2 per diffused over time, with most of the infestation carried pot 12 days after planting (DAP), and finally to 1 plant out at 58 and 62 DAP. The neonate larvae were per pot after the first damage rating. The least produced from egg masses laid on wax paper in the damaged plants were retained during thinning. screen cages in the laboratory by adults derived from Seeds of NK5445 Bt experimental hybrids were field-collected pupae. imported from Syngenta (Hawaii), while seeds of NK5445 non-Bt were imported from Thailand. Evaluation Syngenta (Philippines) supplied the seeds of Sugar 73 Leaf-feeding damage was evaluated using the and NK5468. Plant Quarantine Officers, following Guthrie et al (1960) 1-9 leaf-feeding damage scale as established protocol, supervised importation and shown in Table 1. Individual plants were evaluated 14 subsequent handling of the imported seeds. days after the first infestation, and 11 days and 14 days after the second infestation.

54 Bt Maize Against Asiatic Corn Borer At harvest, damage to the stalks and ear shanks (Figure 1). An ante-room and disinfectant (Lysol) foot- was evaluated by splitting these along the length, and dip were provided. To avoid heat buildup during warm counting the holes and measuring the total length of days, the concrete floor of the plastic house was tunneling by ACB. Damage to the tassel was covered with coco-coir matting that was kept wet by determined by counting the number of cut tassel and drip irrigation. A 50-cm fan aided in air circulation. number of holes in each tassel. Damage to the ear was The prevailing air temperature inside the greenhouse determined by counting the number of holes and was 30-33°C at mid-day. Prevailing soil temperature percent of kernel damaged. at 15 cm depth during mid-day was in the range of 22- 24°C. The entire floor of the plastic house, including Trial facility and plant care the immediate perimeter area were cleaned with The study was conducted under the supervision of pressurized water and subsequently sanitized with the National Committee on Biosafety of the chlorox everyday throughout the study. Philippines (NCBP), the Institutional Biosafety The plastic house was secured by a perimeter Committee of the University of the Philippines in fence of interlinked wire with 3 strands of barbed wire

Figure 1. View of the plastic house at 40 days after planting

Mindanao and the Plant Quarantine Office of the on top. To prevent rodent invasion, the perimeter at Bureau of Plant Industry. The study was conducted in the bottom of the fence down to the soil level was a 15 x 6 x 4 meters (length x width x height) insect- covered with a strip of galvanized metal sheet 50 cm proof screenhouse specially built for the purpose wide. Entrance to the enclosed facility was restricted

Eufemio T Rasco Jr et al 55 to the project personnel and others authorized by the RESULTS Plant Quarantine Service. Steam-sterilized garden soil was used as growing Leaf/plant feeding damage rating medium for the maize. Watering was done daily to Table 2 shows that the 3 experimental hybrids of keep the soil moisture near field capacity. Pots were NK5445 Bt were uniformly rated HR (highly resistant; sited on plastic shallow pails to catch excess water. 1 in a scale of 1-9), showing no visible injury or only a At planting, 45 grams of 14-14-14 granular small amount of pin-hole or fine shot-hole type of fertilizer was applied per pot. Fertilizer application feeding injury on a few leaves. This rating was was repeated at 21 and 32 DAP using 2.5 grams per consistently shown across 3 assessment periods (41, pot of granular urea (45-0-0). The plants were not 52 and 55 days after planting). Within 2 days after egg treated with any kind of pesticide. mass or larvae introduction, dead larvae were Natural pollination was supplemented with observed on the leaves of these entries. In contrast, the artificial pollination using pollen from excess plants of 3 check entries (isogenic hybrid of NK5445 non-Bt, NK5468 that were grown similarly in pots for the sweet corn hybrid and NK5468), showed visible purpose. damage as early as 2 days after artificial infestation (Figure 2). They were rated I (intermediate; rating of Schedule of the study 5.2) to HS (highly susceptible; rating of 8.2), HS The study was conducted starting during the cool showing several elongated lesions on as much as two- part of the year, on 15 December 2002, and thirds of the leaves.

Table 2. Leaf /plant feeding ACB damage rating (Guthrie scale)

Assessment Period1 Entries Early Vegetative Late Vegetative Final (55 DAP)** Stage (41 DAP)** Stage (52 DAP)** NK5445 Bt (A) 1a 1a 1a NK5445 Bt (B) 1a 1a 1a NK5445 Bt (°C) 1a 1a 1a Non-Bt isogenic line of NK5445, check 7c 7.9b 6.9b Sweet corn hybrid (Sugar 73), check 7c 7.5b 8.2c NK5468 (commercial hybrid), check 5.2b 7.4b 7.6bc Mean 3.7 4.3 4.3 CV (%) 23.0 14.8 19.5 **F-test shows that the differences among entries are significant at .01 level. Kruskal-Wallis and Chi-square (non-parametric tests) show similar results. 1Values in the same column followed by the same letter(s) are not significantly different using DMRT.

terminated on 27 March 2003 in the beginning of the Among the 3 experimental hybrid versions of warm and dry months. NK5445 Bt, hybrid version A did not show any damage or abnormality at all. Both experimental Disposal of plant and soil materials hybrids B and C showed minor injury (small holes) All plant materials were burned and their and some stunted plants (unrelated to ACB injury). residues buried after they were used. These included whole plants thinned out, tassels, ears, stalks, and Stalk and ear shank damage grains. The soil used, together with the root residues, During harvest at 102 days after planting, were steam-sterilized for 4 hours before disposal. damage evaluation (Table 3), only minor tunneling

56 Bt Maize Against Asiatic Corn Borer was seen in the stalks of all experimental hybrids of Tassel, ear, and kernel damage NK5445 Bt. Tassel damage was high among the checks, In contrast, the check entries showed a range of showing a range of 50% to 86% of tassel cut near the 9.9 to 13.4 holes per stalk, with a mean length of base, dried and toppled over before harvest (Table 4). feeding tunnels ranging from 350 to 621 mm. ACB Again, the sweet corn hybrid was most susceptible, pupae were observed inside the feeding tunnels. The with the highest percent cut tassel and number of sweet corn hybrid showed the highest degree of holes. In contrast, experimental hybrids A and B of damage. In addition, 30% of the plants of the sweet NK5445 Bt showed no cut tassel at all, while corn hybrid lodged before harvest due to severe injury experimental hybrids B and C showed slight feeding near the base of the stalk. None of the other entries damage (few small holes). showed lodging. Percent ear infested was also high among the In the same manner, the check entries showed checks, showing a range of 54.2 to 69%. Among the damage on the ear shanks, with a maximum of 1.9 experimental hybrids of NK5445 Bt, A and B showed

Figure 2. Typical ACB feeding damage on corn leaf of susceptible entry (0.3 to 0.5 holes per stalk, with maximum length of 4.7 mm). No pupa was observed. Tunneling was absent on the ear shanks.

holes per ear shank and 33.9 mm length of feeding no damage, while C showed minor infestation (3.3% tunnel. infested). The high CVs of stalk and ear shank damage data Unlike the other data, which showed substantial were probably caused by the lack of randomness in the damage caused by ACB, kernel damage was relatively movement of late instar larvae across plants, minor (Table 4). Three ears out of 10 examined of each particularly those that came from later infestations, of Isogenic NK5445 Non-Bt and NK 5468 showed ACB which may have been caused by the early death of feeding at the tip of the cob. However, the trend of 30% of the sweet corn hybrid plants, and differences in kernel damage was consistent with the other data: the growth rate (and consequent differences in degree of checks were much more severely damaged than the 3 foliage overlaps among plants) caused by temperature experimental hybrids of NK5445. gradient from the edge to the center of the plastic High CVs in number of holes in the tassels were house. likely caused by differences in time that the tassels

Eufemio T Rasco Jr et al 57 were cut and dried up after initial damage by the ACB. materials towards the end of the study is consistent The high CVs in ear and kernel damage may have with the published information that shows that: a) been caused by the same factors that caused high CVs concentration of the cry1Ab endotoxin tends to decline in stalk and ear shank damage. In addition, this was with plant age (15-29ug/gram fresh weight for mature probably caused by the highly variable number of ears and young leaves), and b) concentration of the cry1Ab and kernels, upon which the percent infestation was endotoxin is lower in the kernel (5 parts per billion) based. than in the leaves (Essential Biosafety 2002). The minor injuries observed in the Bt hybrids can further Other arthropods observed be explained by infestation of larger larvae coming Starting from the middle of the study period, other from neighboring plants of the susceptible non-Bt species of insects were observed to be infesting the checks. This was possible because of the overlaps in maize plants (Table 5). There was no clear pattern of leaves of neighboring experimental plants. Larger

Table 3. Evaluations of stalk and ear shank damage by the ACB

Stalk Damage1 Ear Shank Damage1 Entries Number of Length of Number of Length of entrance feeding tunnels entrance feeding tunnels holes** (mm)** holes** (mm)** NK5445 Bt (A) 0.5a 4.7a 0a 0a NK5445 Bt (B) 0.3a 3.0a 0a 0a NK5445 Bt (°C) 0.3a 1.0a 0a 0a Non-Bt isogenic line of NK5445, check 10.7b 350b 0.9b 33.9b Sweet corn hybrid (Sugar 73), check 13.4b 621b 1.9c 26.9b NK5468 (commercial hybrid), check 9.9b 406b 0.7b 18.9b Mean 5.88 230.98 0.58 11.49

CV (%) 31.49 29.84 38.25 88.65 **F-test shows that the differences among entries are significant at .01 level. Data on number of entrance holes were transformed to square root of x+0.375. Data on length of feeding tunnels on ear shanks were transformed to square root of x+0.375 with final analysis limited to entries showing some degree of damage. 1Values in the same column followed by the same letter(s) are not significantly different using DMRT incidence; the Bt hybrids were as much affected as the larvae from the susceptible plants, because of their check entries. The fire ants were the first to come, higher body weights, may have been able to cause followed by the corn aphids, then by spider mites and some damage before they succumbed to the cry1Ab corn planthoppers towards the end of the season. endotoxin of the NK5445 Bt. However, they were able Initially the insects were collected using a vacuum to make only small holes and short feeding tunnels at cleaner, but this was stopped, as they continued to worst. Consistently, these larvae failed to pupate in increase and it was difficult to remove all of them. the NK5445 Bt entries. In commercial plantings, it is expected that the DISCUSSION NK5445 Bt entries will perform better because there will be no source of big larvae to cause the damage; The study clearly demonstrates the efficacy of Bt- any neonate larva from egg masses laid by adult ACB 11 in all the 3 NK5445 Bt experimental hybrids from other fields will die as soon as it hatches and against the ACB. This was consistently observed in the starts feeding. vegetative and reproductive stages of the maize The results also suggest that the yield advantage plants. The minor injuries sustained by the Bt consistently shown by Bt maize in field trials

58 Bt Maize Against Asiatic Corn Borer whenever corn borer is a factor, can be obtained not done 3 times during the vegetative stage and again only from reduced damage to the leaves, stalk, ears during harvest at 102 DAP. and kernels but also from reduced damage to the Assessments during the vegetative stage showed tassels. Tassels that are cut or otherwise injured by that the NK5445 Bt experimental hybrids, with little ACB early in their development could fail to supply the or no visible injuries to leaves, were highly resistant to amount of pollens necessary to sustain high yields. ACB (mean rating of 1 in a scale of 1 to 9). In contrast, The presence of 4 other species of insects in Bt and the check entries showed several elongated holes non-Bt entries equally well is an indication of the typical of ACB feeding injury on as much as two-thirds safety of cry1Ab endotoxin on non-target insects. of the leaves. The checks were rated I (intermediate in susceptibility), with a mean rating of 5.2, to HS SUMMARY & CONCLUSIONS (highly susceptible), mean rating of 8.2. At harvest, the checks showed severe damage on Three experimental hybrids of NK5445 Bt hybrid the stalks, ear shanks, ears and tassels and relatively maize developed by Syngenta Seed Company and 3 minor injuries on the kernels. In contrast, damage on

Table 4. Tassel, ear and kernel damage by the ACB1

Entry Tassel damage2 Ear damage2 Kernel damage

% cut No of holes** % Infested** % Infested* NK5445 Bt (A) 0 0a 0a 0a NK5445 Bt (B) 0 0.1a 0a .02a NK5445 Bt (°C) 0.11 0.2a 3.3a 0a Non-Bt isogenic line of NK5445, check 50 1.7b 54.2b 0.33b Sweet corn hybrid (Sugar 73), check 86 3.4b 69.0b 1.64b NK5468 (commercial hybrid), check 60 1.9b 58.2b 2.94b Mean 32.69 1.2 30.78 0.82

CV - 35.49 74.02 201.06 1Data for number of holes were transformed to square root of x+0.375; data for fraction infested were transformed to arcsine x where zero values were substituted by 1/4n and 1 values were substituted by 1-1/4n, where n is the number upon which % data was based. Final analysis was based on data from entries that showed some degree of damage. **F-test shows that the differences among entries are significant at .01 level; *F-test shows that the differences among entries are significant at .05 level. 2Values in the same column followed by the same letter(s) are not significantly different using DMRT

checks (non-Bt isogenic hybrid of NK5445, sweet corn the NK5445 Bt entries was zero to minimal. The hybrid, and NK5468) were tested in the screenhouse minimal damage could have been caused by the against the Asian corn borer (ACB) (Ostrinia infestation by larger larvae that came from furnacalis) to evaluate the efficacy of Bt-11 containing neighboring susceptible plants. the truncated gene cry1Ab. The experiment was set up The study clearly demonstrated the efficacy of the in a completely randomized design with 10 cry1Ab protein present in Bt-11 maize in controlling replications, each represented by a 12-inch pot with a ACB under screenhouse conditions. This advantage final population of 1 plant per pot. The experimental can be greater under commercial planting in the field plants were repeatedly infested with egg masses of where the insect-protected plants will not be mixed ACB at 27 days after planting (DAP), then again at 41 with susceptible plants. The presence of 4 other insect DAP. The tassels and silks were also infested with species in both checks and NK5445 Bt plants, possibly larvae at 55-69 DAP. Feeding injury assessments were introduced through the field-collected ACB egg masses,

Eufemio T Rasco Jr et al 59 is an indication that cry1Ab poses no threat to non- target insects, it is therefore recommended that open target insects. field testing be conducted to verify the efficacy of Bt11 Based on the clear efficacy of Bt-11 against ACB as well as the stability of the control under natural and the observation that it did not pose threat to non- and wider range of environments.

Table 5. Other arthropods observed

Common Name Scientific Name Corn aphid Rophalosiphum maidis Corn planthopper Peregrinus maidis Red spider mites Tetranychus sp. Fire ants Solenopsis geminta

LITERATURE CITED

Essential Biosafety (CD). 2002. Agbios, Merrickville, Ontario, Canada Fernandez EC, SC Dalmacio & RA Hautea. 1997. Efficacy of transgenic corn expressing the Bacillus thuringiensis cryIA(b) gene against the Asian corn borer, Ostrinia furnacalis (Guenee). Philippine Entomologist 11(2): 119-130 Guthrie WD, FF Dicke & CR Neiswander. 1960. Leaf and sheath feeding resistance to the European corn borer in eight inbred lines of dent corn. Ohio Agricultural Experiment Station Research Bulletin 860 James C. 2005. Preview: Global status of commercialized transgenic crops: 2004. ISAAA Briefs No 32-2004. www.isaaa.org February 17, 2005 Phipps RH & JR Park. 2002. Environmental benefits of genetically modified crops: Global and European perspectives on their ability to reduce pesticide use. Journal of Animal and Feed Sciences 11: 1-18 Yorobe JM Jr. 2004. Economic impact of Bt corn in the Philippines. Paper presented during the 45th National Convention of PAEDA, October 2005, Quezon City, Philippines

60 Bt Maize Against Asiatic Corn Borer