Two types of bioassays, in petri dishes and in pots, were conducted to determine if rootworm resistance could be imparted to maize via a bridge species derived from crossing Tripsacum dactyloides and Zea diploperennis. For the bioassays, 1,000 non-diapausing western corn rootworm, Diabrotica virgifera, eggs in soil were shipped from French Agricultural Research, Inc., Lamberton, Minnesota, to Durham, NC, under U. S. Department of Agriculture permit number 922762. Plants were infested with newly hatched first instar larvae of western corn rootworm. The larvae were transferred to test containers by lifting with a small paint brush. Two separate petri dish bioassays and three pot bioassays were performed.
For the bioassays, seed from Tripsacorn, i.e. Tripsacum dactyloides (female) x Zea diploperennis was crossed with four diverse types of maize. The four maize types included: Funk's G4522, a commercial hybrid corn seed; B73 and W64A, two inbred lines; Zapalote Chico, a native Mexican race classified as a prehistoric mestizo derived from ancient indigenous races. Other plants infested with corn rootworm included G4522 x Sun Dance, Tripsacum, Tripsacorn and maize controls. Sun Dance refers to Zea diploperennis (female) x Tripsacum dactyloides.
Petri dish bioassays were employed to screen for antibiosis versus antixenosis. If there is an antibiotic effect, evidence for eating and dead larvae can be seen; if there is an antixenotic effect, larvae can be observed trying to leave the dish. For these tests, 10 grams of top soil sieved through a 1mm mesh screen was placed in a petri dish with 3 to 5 freshly germinated seedlings or, in the case of Tripsacum, with a small clonal piece of plant with young roots, and kept moist. The rims of each dish were ringed with petroleum jelly to monitor for any larvae trying to leave the dish. Up to a total of 50 larvae were added to each dish over a three day period. Each treated dish was observed for several days under a dissecting microscope at 60X magnification and behavior recorded.
Results of petri dish bioassays are summarized in Table 1. In all cases, larvae remained on or near the roots, seed and cotyledons, or in the soil. There was no indication of larvae trying to exit dishes; thus, there is no evidence for antixenosis. Tripsacum, Tripsacorn, B73 x Tripsacorn, W64A x Tripsacorn, G4522 x Tripsacorn and G4522 x Sun Dance did not show any signs that the roots produce a substance that is a deterrent to the insects. Evidence for antibiosis and tolerance was indicated with Tripsacorn and the hybrids between corn and Tripsacorn tested, whereas there was no evidence for antibiosis or tolerance with the corn and maize x Sun Dance materials tested.
Plants grown in pots were used to screen for evidence of tolerance and/or antibiosis. Lodging is seen in plants that are susceptible to rootworm damage, whereas plants that remain upright and healthy when exposed to rootworms are indicative of tolerance and antibiosis. Root damage was observed and scored according to the Hills and Peters rating scale from 1 to 6 that is widely used in the corn belt to evaluate root damage (J. Econ. Entomol. 64:764-765, 1971). The rating criteria are: (1) no damage or only a few minor feeding scars; (2) feeding scars evident but no roots eaten off to within 1 1/2 inches of the plant; (3) several roots eaten off to within 1 1/2 inches of the plant but never the equivalent of an entire node of roots destroyed; (4) one root node completely destroyed; (5) two root nodes completely destroyed; (6) three or more root nodes destroyed.
Table 1. Results of petri dish bioassays.
| No. of Larvae | Observations/Comments | |
| Bioassay #1 | ||
| Tripsacum | 50 | Larvae stay on root, some feeding but virtually no damage to roots, larvae not visible after a couple of days |
| Tripsacorn | 50 | Some feeding, little root damage |
| B73 x Tripsacorn | 50 | Some feeding, little root damage, plants continue to grow |
| G4522 x Tripsacorn | 50 | Some feeding, little root damage, plants continue to grow |
| G4522 x Sun Dance | 50 | Extensive feeding, plants died |
| Corn control | 50 | Extensive feeding, plants died |
| Bioassay #2 | ||
| Tripsacorn | 20 | Light feeding, some dead larvae |
| Corn control (W64A) | 45 | Extensive feeding, plants died |
| W64A x Tripsacorn | 45 | Feeding on roots, seed and cotyledons, some dead larvae |
When a bioassay was complete, two or three plants were removed from pots, soaked in water, then rinsed with a gentle spray to thoroughly clean roots, and observed under a dissecting microscope for scoring. The mean calculated from the total scores of plants in each category is reported. Tolerant plants may suffer root damage but are capable of regrowth and varying degrees of plant recovery. Well developed secondary root systems are often capable of compensatory growth from damaged crown roots.
In the first pot bioassay, 3 to 5 seedlings (approximately 1 week old), or in the case of Tripsacum a small clone with young roots, were planted in potting soil in 10-ounce containers and were grown indoors under artificial grow lights. A total of 70 larvae were added to each container over a two day period and plants were observed for 11 days.
In the second pot bioassay, ?10 day old seedlings were planted in potting soil in 3 inch peat pots and grown indoors under artificial grow lights. A total of 30 larvae were added to each pot over a three day period. Although most plants were dead within one week, observation of the ones that survived extended over two weeks before plants were sacrificed for root evaluation. For each type, there were a minimum of two plants, and in most cases there were four plants.
In the third pot bioassay, plants were grown under natural light and were 11 to 14 days old at infestation. A total of 30 larvae were added to each pot over two days. Observation was for 11 days before sacrificing plants to score root damage.
Results of pot bioassays are summarized in Table 2. The data indicate that maize x Tripsacorn plants are definitely more resistant to corn rootworm than corn controls and maize x Sun Dance. Antibiosis and tolerance are evidently the mechanisms for resistance inherited from Tripsacorn. All plants sustained some injury to roots. Lodging in corn controls and maize x Sun Dance plants was >45o and damage on the Hills and Peters scale ranged from 5 to 6. Although maize x Tripsacorn plants sustained some root damage, they remained upright and appeared healthy. There was good development of secondary roots from the damaged crown showing capability for compensatory growth in all maize x Tripsacorn plants examined. One plant each of Zapalote Chico x Tripsacorn and W64A x Tripsacorn from Bioassay #3 was replanted after examination for root damage. Both have completed the growth cycle and produced seed.
Table 2. Results of pot bioassays.
| No. of Larvae | Duration | Root Damage* | Observations | |
| Bioassay #1 | ||||
| Tripsacum | 70 | 11 days | Not recorded | No sign of damage |
| Tripsacorn | 70 | 11 days | Not recorded | No sign of damage |
| B73 x Tripsacorn | 70 | 11 days | Not recorded | No sign of damage |
| G4522 x Tripsacorn | 70 | 11 days | Not recorded | No sign of damage |
| G4522 x Sun Dance | 70 | 11 days | Not recorded | Plants died at 6 days |
| Bioassay #2 | ||||
| Corn control (W64A) | 30 | 14 days | 5.0 | Plants died |
| W64A x Tripsacorn | 30 | 14 days | 2.0 | Plants weakened |
| G4522 x Sun Dance | 30 | 14 days | 5.0 | Plants died |
| Bioassay #3 | ||||
| Corn control (Zapalote Chico) | 30 | 11 days | 4.0 | Lodging (>45o), leaf damage |
| Zapalote Chico x Tripsacorn | 30 | 11 days | 2.0 | Minor leaf damage |
| Corn control (W64A) | 30 | 11 days | 4.0 | Lodging (>45o) |
| W64A x Tripsacorn | 30 | 11 days | 2.3 | Plant upright, growing |
| G4522 x Sun Dance | 30 | 11 days | 5.0 | Lodging (>45o), leaf damage |
| Tripsacorn | 30 | 11 days | 1.0 | No damage |
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