Relationships of Helicoverpa armigera, Ostrinia nubilalis and Fusarium verticillioides on MON 810 Maize

MON 810 maize was developed against Ostrinia nubilalis and is suggested to indirectly decrease Fusarium spp. infestation in maize ears. To evaluate this effect, co-occurrence of insect and fungal pests on MON 810 maize was studied. During 2009, exceptionally high maize ear infestation occurred in Julianna-major (Hungary). From investigation of some thousands of maize ears, the majority of the larval damage originated from Helicoverpa armigera larvae, while O. nubilalis larvae contributed significant damage only at a single plot. Fusarium verticillioides infection appeared only in a small portion (∼20–30%) of the insect damaged cobs. H. armigera and O. nubilalis larvae feeding on F. verticillioides mycelia can distribute its conidia with their fecal pellets. MON 810 maize showed 100% efficacy against O. nubilalis in the stem, but lower efficacy against O. nubilalis and H. armigera in maize ears. The ∼Cry1Ab toxin content of maize silk, the entry site of H. armigera, was lower than that in the leaves/stem/husk leaves of MON 810. Fusarium-infected MON 810 cobs are rarely found and only after larval damage by O. nubilalis. H. armigera larvae could not tolerate well F. verticillioides infected food and attempted to move out from the infected cobs. For further feeding they re-entered the maize ears through the 8–12 husk leaves, but in the case of the MON 810 variety, they usually could not reach the kernels. Apical damage on cobs resulted in only a minor (about one-tenth of the cob) decrease in yield.


Introduction
MON 810 maize, expressing a transgene encoding a truncated version of the lepidopteran-specific Cry1Ab toxin of Bacillus thuringiensis var. kurstaki [1], was developed against Ostrinia nubilalis (Hübner) (Lep., Crambidae) and is suggested to indirectly decrease Fusarium spp. infestation [2,3] resulting in decreased mycotoxin content in the genetically modified (GM) crop [3][4][5]. Different additional lepidopteran pests of maize occur in the American continents and in Europe, i.e. Helicoverpa zea (Boddie) (Lep., Noctuidae) in North America, Spodoptera frugiperda (J. E. Smith) (Lep., Noctuidae) in South America, Helicoverpa armigera (Hübner) (Lep., Noctuidae) in Eastern Europe and Sesamia nonagrioides (Lefebvre) (Lep., Noctuidae) in Western Europe [3,6]. O. nubilalis is a stem and maize ear pest, while larvae of European noctuid species damage only maize ears. In the Pannonian Region, where maize ear damage mostly originates from O. nubilalis and H. armigera, strong infestation by O. nubilalis occurs only about once every ten years [7]. In the ongoing experimental cultivation series at Julianna-major (the Ecological Research Station, Plant Protection Institute), where maize has been cultivated in several plots during the last decade, significant infection caused by H. armigera and O. nubilalis larvae occurred only in 2009. Presently, after soil-disinfection pesticides (formerly used against Melolonthidae and Elateridae larvae) have been withdrawn, Hungarian farmers do not use chemical insecticides in maize production. The low frequency of maize ear damage caused by lepidopterous larvae may question the economic usefulness of MON 810 maize in this region [7]. Presently, the owner of the genetic event offer MON 810 maize varieties with the rational that lower larval damage of maize ears may reduce Fusarium spp. infestation, therefore, fusariotoxin contamination of such kernels is lower. A certain professional debate exists in this field: some groups claim that maize ear rot is associated with feeding damage caused by O. nubilalis and H. zea larvae in the U.S. [3], others found this association apparent in the case of O. nubilalis only [4]. The aim of this study was to clarify such pest co-occurrence in Hungary, one of the greatest maize producers in Europe.

Stock Colonies
Potato dextrose agar was used for stock culture of F. verticillioides originating from a single microconidium (storage: 10 °C ) [8]. Laboratory stock colonies of O. nubilalis and H. armigera were established. For a stock colony of O. nubilalis, adults were collected during 2004 in Ké ty (Hungary). Larvae were fed on a semi-synthetic diet [9]. For breeding of H. armigera, larvae were collected during 2008 in Zsá mbé k (Hungary). Larvae were fed on a different semi-synthetic diet [10].
Lepidopteran larvae reared for the field experiments in maize ear isolators were fed on sweet maize (Jubilee) discs with or without F. verticillioides mycelia. To prepare a F. verticillioides infected disc, sweet maize cobs were cut to 3 cm thick discs. Discs were dipped into an aqueous solution (200 mL) including a stock culture of F. verticillioides and 0.1% Tween 20. The final concentration was ~80,000 microconidia/mm 3 . After four days (incubation: 22-25 °C ), when the distribution of mycelia was at least ~50% on the surface of a single disc, second or third instar larvae were transferred onto them. Larvae were fed on sweet maize discs with or without F. verticillioides mycelia until the next molt.

Work in Maize Ear Isolators
Field work was done at the Ecological Research Station, Plant Protection Institute at Julianna-major (the north-western outskirts of Budapest). Maize ear isolators were placed on MON 810 or near isogenic plants at the beginning of silking time (August 13, 2009). Isolators were placed on artificially infected cobs using third or fourth larval stadia. The stem was cut above the maize ear at the height of maize silk, an appropriately treated larva was placed on it, and the fine mesh maize ear isolator (diameter = 10 cm, length = 35 cm) was placed on the entire maize ear and was tightly closed at the bottom. Treatments were: (i) H. armigera larvae having consumed F. verticillioides; (ii) H. armigera larvae not having consumed F. verticillioides; (iii) O. nubilalis larvae having consumed F. verticillioides; (iv) O. nubilalis larvae not having consumed F. verticillioides mycelia; and (v) untreated control (Table 1). Repetitions were 10-14 for H. armigera and 18-20 for O. nubilalis. A treated control (vi; 20 repetitions) consisting of F. verticillioides mycelia on RbGUM media disc (5 mm diameter) fixed on maize silk was also used. After two weeks, maize ear isolators were collected and their content was carefully checked.

Measurement of Cry1Ab Toxin Content in Maize Ear Parts of MON 810
Cry1Ab toxin content in the main parts of the maize ears was determined by a commercial sandwich immunoassay, Abraxis Bt-Cry1Ab/Ac ELISA kit (#PN 51001, Warminster, PA, USA) carried out in 96-well microplates according to manufacturer-provided protocol. ELISA signals were detected on an iEMS microtiter plate reader (Labsystems, Helsinki, Finland). Cry1Ab protoxin calibrators (Abraxis) were put on every microplate at concentrations between 0.25 and 4 ng/mL, assays were used for determination of analyte concentration by linear regression. Activated Cry1Ab toxin concentrations were calculated from detected protoxin concentration values with Cry1Ab activated toxin/protoxin cross-reactivity, 56.4%, previously determined for the Abraxis Bt-Cry1Ab/Ac ELISA kit [11,12].

Work under Field Conditions
A MON 810 (DK-440 BTY) and its near isogenic line (DK-440) were investigated during October 5-8, 2009, in Julianna-major. Depending on the plot size, some hundreds (ca. 100-400) of maize ears were collected from every assortment. Upon removal of husk leaves, cobs were carefully investigated for symptoms and severity of larval and fungal damage (proportion of the damaged part in the cob, location of the damage, occurrence of pink ear rot as a sign of fungal infection). Four replicates were used resulting in 457 to 1,339 cobs investigated per a single plot. Data without transformation were analyzed using Statistica ver. 5.5 program (ANOVA and Tukey test). Insect and plant samples were also collected for Fusarium spp. identification in the laboratory.
To evaluate the real yield loss, cobs (10 replicates) in different sizes (small, medium and big size of cob) infected by H. armigera larva were chosen. The infected part of the cobs was removed and masses were measured.

Fusarium verticillioides Transmission Caused by Individually Isolated Larvae
Fusarium verticillioides was identified in laboratory fungal rearing tests (using Fusarium-selective RbGUM media) from plant and fecal pellet samples collected by the maize cob isolators in the field experiments. Thus, F. verticillioides infection was verified in fecal pellets of H. armigera and O. nubilalis, husk leaves, ripening kernels and cobs from maize plants damaged by H. armigera and O. nubilalis, as well as stem tunnels caused by O. nubilalis. Insect-transmitted infestation of F. verticillioides (manifested in the occurrence of pink ear rot) appeared to be dependent on the choice of the plant part by the insect to feed on, and on the varying subsequent survival rates due to different Cry1Ab toxin exposures. In contrast, no infestation by F. verticillioides was observed in the untreated control, and none was produced by fixing F. verticillioides mycelia on corn silk, either ( Table 1). All the H. armigera larvae used (third and fourth stadia) chose maize ears for feeding. The majority of O. nubilalis larvae (third and fourth stadia) preferred to feed on the fresh wound on the stem, where it had been cut prior to the placement of the maize ear isolators on isogenic maize. A small proportion (10-15%) of O. nubilalis larvae tended to feed on husk leaves on MON 810 maize ( Table 1). Larvae of both species investigated, having been fed previously in laboratory rearing during one larval instar on F. verticilliodes mycelia, showed increased mortality (10-17%). F. verticillioides infection was transmitted only by larvae having been fed previously on its mycelia (Table 1). Nonetheless, not all larvae (H. armigera and O. nubilalis) having been fed on F. verticillioides mycelia could transmit Fusarium infestation; the rate was only 20-42%. All of the O. nubilalis larvae died on MON 810 maize, although only this species could transmit F. verticillioides infection feeding on the base of maize ears. Some H. armigera larvae (third and fourth stadia) could survive on MON 810 maize, feeding on husk leaves (Table 1). These larvae stop feeding from time to time, and starve-showing the symptoms of Cry1-toxicosis-consuming the minimum possible.

Helicoverpa armigera, Ostrinia nubilalis and Fusarium verticillioides Infection under Field Conditions
Similarly high levels of maize ear infestation (42-46%) were observed in all three, closely located maize fields investigated. Only 20-30% of cobs with larval damage were also infected by F. verticillioides. Larval damages were mostly attributed to H. armigera in two of the maize fields, while both H. armigera and O. nubilalis larvae occurred in nearly identical rates in the third case ( Table 2). Independently from the damaging insect species, mostly apical cob infestation occurred. Fusarium verticillioides infestation was significantly higher in one case, but it did not correlate with the overall larval damages ( Table 2).
In case of O. nubilalis, the tunnels were found mostly on stems, and only a quarter of the damage occurred on maize ears ( Table 3).
The position where the larvae fed on the maize ears were different for the two species evaluated. In the case of H. armigera, ~90% of the damage occurred on the apical region. A lower value (~80%) of apical damage was related to O. nubilalis (Table 4). First instar larvae of both species usually try to reach the kernels through the maize silks, although a more significant portion of O. nubilalis larvae choose the base of maize ear. In case of microbial infection (like F. verticillioides), older H. armigera larvae might move (~10%) toward the middle of the cob, seeking a drier environment, or come out from the maize ear at the top and choose another maize ear. In this latter case, older H. armigera larvae choose husk leaves to reach the middle of the cob. The most frequent damage type by H. armigera larvae occurs on the top of the cob by feeding on ripening seeds. An ample amount of fecal pellets may be found at places of earlier seeds. Fecal pellets of H. armigera are sometimes covered by mycelia of different fungi. In our case, plant pathogenic F. verticillioides was the most abundant ( Table 5). The damage type caused by O. nubilalis larva is different. It makes a longer tunnel toward the base of the cob under the seed surface. Fecal pellets of O. nubilalis are usually not visible on the cob surface, thus damage is not so bulky.   The ~Cry1Ab toxin content (corrected with toxin/protoxin cross-reactivity [11,12]) was found to be 826 ng (s.d. = 237); 1280 ng (s.d. = 150) and 2075 ng (s.d. = 1287) ~CryAb toxin/g fresh mass in corn silk; husk leaves and young cob, respectively. The high standard deviation in the cob is most likely due to this plant part being a mixture of different tissues with variable amounts of ~Cry1Ab toxin.
Apical infection by H. armigera larvae-the most frequent damage type (Table 4)-results in the loss of only 10-15% of the entire cob (Table 6). Infection and damage on the base and the middle of the cobs might cause more damage.

Effect of MON 810 on Helicoverpa armigera under Field Conditions
MON 810 maize shows a high efficacy against H. armigera and O. nubilalis larval infection. None of the O. nubilalis larvae survived in stems, but some in maize ears (Table 7). There are some survivors in the case of H. armigera in maize ears. MON 810 drastically reduced F. verticillioides infection as well.

Lepidoptera Larvae and Fusarium verticillioides Mycelia
It was apparent from the laboratory experiments that young larvae of H. armigera and O. nubilalis do not tolerate well F. verticillioides mycelia in their food (Table 1). In a laboratory experiment, H. armigera L1 could develop only until the third larval stadium (data not shown). O. nubilalis larvae could tolerate F. verticillioides mycelia much better. Nonetheless, H. armigera and O. nubilalis larvae feeding on F. verticillioides mycelia for a short period can spread fungal microconidia, which can survive the digestive channel of these insects. F. verticillioides was readily identified from the fecal pellets of both species. H. armigera larvae usually attempt to escape from moldy tunnels. During this period, the larva can transmit F. verticillioides. Evaluating co-occurrence of insect and fungal infection on the isogenic maize line, only 20-40% of the larva feeding on F. verticillioides infected seeds was found to transmit maize pink ear rot (Table 1). Similarly, some 20-30% of larval infection was found to be related to F. verticillioides infection in the field experiments ( Table 2). In contrast, MON 810 maize, highly effective against the larvae, strongly reduced the F. verticillioides infection as well (Table 7). Incidental wounds on the top of maize ears [13] may play a more important role in this relationship than direct transmission. Insecticide (lambda-cyhalothrin) application against O. nubilalis, monoculture and effects of sowing date also indicate this effect [14][15][16][17][18]. In agreement with reported results [19,20], infestation through intact maize silk with F. verticillioides did not occur. Certain, but not all Fusarium mycotoxins were found to be related to O. nubilalis damage on Bt-plants (SYN-EV176 and MON 810 events) [21].

Effectivity of MON 810 Maize
MON 810 maize is advertised to be used against O. nubilalis larvae, which usually feed on the base of the leaves after hatching and subsequently make a tunnel into the stem (Table 4). MON 810 maize leaves produce the largest concentration and amount of the truncated toxin (ca. 10,000 ng ~CryAb toxin/g fresh mass), while only a moderate concentration (ca. 1,500 ng ~CryAb toxin/g fresh mass) is produced in the stem [11,12]. Different parts of the maize ear produce moderate (husk leaves, cobs) or low (maize silk) levels of ~Cry1Ab toxin, therefore, are more suitable plant parts for the survival of those larvae which can feed on it. This is the probable reason why first instar larvae usually survive in the upper part of the maize ear feeding first on the maize silk. Similarly to reported results [22], no survival of O. nubilalis larvae was seen in the stem of MON 810 (Tables 1 and 7), but several survived in the lower part of the cob creating tunnels in the base of the husk leaves [23,24]. Larvae surviving in ~Cry1Ab toxin containing maize ear with variable toxin content may become the source of Cry1-resistant strains in the future [25,26]. This applies especially to spots near the rescue zone, where intraspecific hybrid seeds with only one transgenic parental line and, consequently, lower Cry1Ab toxin production levels than the parental GMO trait (data not shown), are frequent.

Yield Loss is Caused by Helicoverpa armigera, Ostrinia nubilalis and Fusarium verticillioides
Although the most frequent apical damage of maize ear is very spectacular (Tables 4 and 5), the yield loss is rather moderate. High cob damage (40-50%) caused by H. armigera and/or O. nubilalis larvae is very rare in Hungary, and the yield loss is only 4-8% of the cob mass even in those infrequent cases (corresponding to 10-16% cob mass, Table 6). In reality, yield losses due to infected cob tops are even smaller, as seeds on the thinner top part of the cobs are often lost anyway during mechanic shelling of cobs. As the fungal infection occurs predominantly apically on the cobs, such loss of cob tops also reduces the average fusarotoxin content in the shelled kernels. This is the basic reason why Hungarian farmers practically do not use chemical insecticides against H. armigera and O. nubilalis, even though authorized preparations are available [27].

Conclusions
Laboratory experiments using third and fourth larval stages of O. nubilalis and H. armigera revealed that: (i) ~Cry1Ab toxin distribution in MON 810 plants modifies survivorship of a single larva depending on where it attempts to enter the plant; (ii) O. nubilalis larvae prefer feeding in stems, while H. armigera larvae feed on corn ears only.
Field experiments with natural infestation indicated: (iii) H. armigera larvae tend to change feeding place in case of F. verticillioides infection, when they attempt to reach kernels via husk leaves; (iv) early infestation caused mostly apical maize ear damage; (v) only in 20-30% of cases was larval damage followed by a F. verticillioides infection; (vi) eventual yield loss is only about one-tenth of the corn ear apical infection; (vii) MON 810 maize effectively controls maize ear infection by H. armigera and O. nubilalis, but some larvae may survive leading to faster development of Cry1Ab-resistance in the future.