8. Thesis structure
3.2 Materials and methods
3.2.3 Bruchid resistance testing
A no-choice test was conducted in the laboratory by infesting dry bean samples in closed jars with bruchids. A free-choice test was done by infesting bean genotypes in the open field. In the no-choice test, the insects were allowed to oviposit and develop on the bean samples provided. In the field experiment, insects could feed and/or oviposit on the bean genotypes of their choice. These experiments were conducted as follows:
(a) Laboratory infestation (no-choice test)
Most genotypes did not yield sufficient seed for screening against both bruchid species, hence genotypes were tested for resistance against either Z. subfasciatus or A.
obtectus, in independent experiments. However, a few genotypes, such as Maluwa and KK35, produced enough seed for testing against both bruchid species.
The first set of 42 genotypes (Appendix 1) was screened for resistance to Z.
subfasciatus in the first experiment, and the second set with a total of 93 genotypes (56 landraces and 33 improved varieties) (Appendix 2 and 3, respectively) were tested for resistance to A. obtectus, in two separate experiments. The SMARC lines, SMARC 2 and SMARC 4, were tested as resistant checks against Z. subfasciatus only because
there was not enough seed to test them against both bruchid species. SMARC lines contain arcelin, which is known to confer resistance against Z. subfasciatus.
Prior to commencement of the test, seed samples were disinfested by freezing at -20oC (Horber, 1989) for at least 10 d. This was done to ensure that any eggs or adult insects from the field were killed. Seed samples were then removed from the deep freezer and were placed in the CTH room (CTH conditions as already described in 3.2.2) for conditioning for 7 d. Seed samples weighing 20± g (approximately 20-25 seeds depending on seed size) were then placed in transparent plastic bottles (Fig 3.2). To prevent the bruchids from escaping, a muslin cloth was used to cover the mouth of each bottle and held in place by a rubber band. Transparent bottles allowed light to enter and allowed ease of observation on a daily basis. The bottles were labelled clearly indicating genotype name, replication number, the date the experiment began and dates of the daily sieving and adult emergence counts.
Figure 3.2. Bean genotypes in transparent bottles placed in the CTH room In all three experiments, the bean samples in each bottle were infested with 10 randomly selected adult insects of 1-3 d old, but without sex determination. Bottles were laid out in a randomised complete block design with four replications on the shelves in the CTH room. Each shelf was large enough to contain a complete replicate. Bruchids were allowed to oviposit on the bean samples for 10 d, after which they were removed and discarded. The samples were further incubated in the CTH room. The adults A. obtectus and Z. subfasciatus started to emerge 28 d and 21 d after the oviposition period,
respectively. The bruchids that emerged were removed by sieving. The bruchids were counted daily until no more emerged (indicating the end of the first generation) at roughly 40 d after infestation.
(b) Field infestation (free-choice test)
A total of 88 bean genotypes were grown in the field at Bembeke experimental site in Dedza district in Malawi during the 2004/05 summer. The two resistant check lines, SMARC 2 and SMARC 4, failed to establish in the field because of poor adaptability to the climate. The experiment was established by planting two seeds per station spaced at 200 mm apart, with 2 ridges spaced at 750 mm apart and plot size was 4 rows x 4 m long with 2 border rows. The net plot comprised 2 middle rows of 3.5 m i.e., 0,25 m on each side were omitted during data collection. Ten genotypes (KK71, KK37, KK01, Nagaga, Maluwa, KK39, KK72, KK87, B91 and KK18) with varying maturity, flower colour, plant height and growth habits were mixed to make a composite variety. This composite variety was used as an infestor row and was planted in a staggered pattern around the experimental field and between test plots, at 3 d intervals, to ensure a steady supply of bruchids to attack the test genotypes (Fig 3.3). Due to limited availability of seed in this first cycle of field screening, the plots were not replicated.
Field layout
Infestor rows
Figure 3.3: Field layout showing infestor rows between plots and around the experimental field
Plot 1
Plot 4
Plot 7
Plot 2 Plot 3
Plot 5 Plot 6
Plot 8 Plot n..
The two bruchid species were collected from farmers’ stores together with infested beans in villages around Bembeke experimental station. Bruchids were collected a day before infestation was done. The bruchids comprised of mixed age adults.
Approximately ±500 g of beans, were collected on which the insects were held temporarily. The bruchids were then carefully counted and placed into vials, ready for artificial infestation in the field.
Bruchids were physically introduced in the field on three occassions; at 100% flowering, full podding and 95% physiological maturity of the bean crop. Fourty insects, consisting of 20 Acanthoscelides obtectus and 20 Zabrotes subfasciatus, were introduced in the infestor rows and at the centre of each plot each time.
For uniformity, each accession was harvested when fairly dry, 1 wk after attaining 95%
physiological maturity. Harvesting all genotypes simultaneously was important to ensure that the beans were given the same length of exposure to bruchid infestation in the field.
The harvested beans, in pods (unshelled), were kept in 10 kg jute twine bags and placed in the CTH room for 60 d at 27-30oC and 70-80% RH, during which time the progeny insects developed. The samples were checked daily to observe any insect emerging.
The counting of emerged adult bruchids was done on a daily basis, after first emergence of bruchids, and until no more insects emerged at 120 d after incubation.