Improved assay procedure for budworm resistance in chickpea
Background
Budworm (pod borer), Helicoverpa armigera and H. punctigera, are major pests in a range of
crops across farmland in Australia. Queensland and northern New South Wales are the worst
affected regions, but growers in most states experience significant damage to a range of crops
including horticultural, fibre, oilseeds and pulses. Chickpea in particular is a preferred crop for
budworm and the crop is affected throughout Australia to some extent. In chickpea, the eggs are
laid by the adult moth on leaves and young pods, where they hatch and the young larvae graze
and develop. The larvae then bore into the pods and destroy the seed. As well as damage to seed,
plants can be completely defoliated during severe infestation if the insects are not controlled by
insecticide spray.
Host plant resistance is an important factor in the control of any insect pest. High levels of
resistance to insects are rare in most plant species, but even partial resistance would play an
important role in an overall pest management package if identified in chickpea.
In our study we measured host plant resistance to budworm indirectly, by examining antibiosis
effects on the insect in a range of cultivars and later in segregating breeding lines. Antibiosis
affects the duration of development of the insect life cycle, vigour and insect survival. Plant cuttings,
from glasshouse grown chickpea plants, were used to feed the larvae. We estimated the effects of
antibiosis in terms of weight of the larvae and noted when diapause began.
Materials and methods
Bioassays with cuttings (excised branches) were done using laboratory cultures of H. punctigera.
An F3 population of chickpea plants, derived from a cross between WAD179 and Annigeri, was
screened for host plant resistance. WAD179 was reputed to be resistant to H. armigera in trials at
ICRISAT, while Annigeri is a susceptible cultivar from India. Chickpea plants were grown in a
controlled temperature glasshouse (22/18°C) until flower initials were observed. Ten replicate
plants were grown in the case of cultivars or advanced breeding lines. Five F3 plants from each F2
were grown, but data was collected from each plant individually since segregation for resistance
was likely within this population. A branch with ten fully expanded leaves was cut from each
plant. Flowers and buds were removed. The cutting was placed inside a perforated plastic bag
(type used in supermarket for French stick bread). One neonate larvae was placed on an upper
leaf. The bag was secured within a fold of foam and held upright in a slot cut into a custom made
lid which fitted over a 200mm black standard pot without drainage holes (figure 1). The pot was
filled with ½ strength Hoagland’s nutrient solution, which was replaced every 2 weeks. Pots were
topped up with water as required when levels dropped due to evaporation and transpiration. The
cuttings in nutrient solution were maintained in a controlled environment room at 20°C; 12 h light
at 400µEm-2s-1.
Larvae were weighted at 14, 21, 28 and 35 days and the cutting was replaced with a new branch
from the same plant. In some cases the larvae died at the neonate stage, and we discarded the
sample and started again. In most cases the neonate progressed to the second instar within 7 days.
We took the weight of the neonate on day one as zero. After 21 days, when some larvae were very
large, we replaced the plant material with two branches instead of one in order to avoid a limited
food source in any sample. Mother plants were maintained in the glasshouse after this time and
allowed to re-shoot, flower and produce seeds for maintenance of the breeding lines.
Figure 1. A cutting method was used to screen for host plant resistance by estimating antibiosis
effects. Cuttings from individual chickpea plants were fed to budworm larvae. Cuttings were
maintained in a fresh state in buckets of nutrient solution in a controlled environment, and were
replaced each week.
Cumulative larvae weights, individual plants
Accession number
179_1
ann_1
65_4
65_3
65_2
65_1
60_4
60_3
60_2
60_1
58_4
58_3
58_2
58_1
56_5
56_4
56_3
56_2
56_1
54_5
54_4
54_3
54_2
54_1
50_5
50_4
50_3
50_2
50_1
48_5
48_4
48_3
48_2
48_1
46_5
46_4
46_3
46_2
46_1
41_5
41_4
41_3
41_2
41_1
-300
-200
-100
0
100
200
300
400
day 14
day 21
day 28
day 35
500
Larval weight (mg)
Figure 2. Resistance of parents and F3 progeny to Helicoverpa punctigera (budworm) screened
using the cutting method to feed larvae and estimate larval weight gain under controlled
environmental conditions over 35 days. WAD179 = ‘179’; Annigeri = ‘ann’ are shown at top of
graph, while the remaining samples are F3 progeny.
Results
The parent line from ICRISAT (179) which was partially resistant to budworm showed slow
weight gain in larvae and slow development from one instar phase to the next. Susceptible
cultivar Annigeri (ann) resulted in faster weight gain in larvae for 28 days, followed by weight
loss as the pupal stage developed before day 35, compared to ‘179’. This corresponded well with
results from field screening in previous studies in India and WA.
The progeny of a cross between these two parents showed a wide range of larval weights; with
some apparently more resistant than WAD179 showing larval weights less than 200mg after 4
weeks. Some progeny were more susceptible than Annigeri. The larvae on these cuttings entered
diapause and pupae formed within 4 weeks.
Ten F3 plants were selected from the group above (fig. 2), on which larvae did not thrive in the
assay. The assay was easily repeated until all of the F3 population was screened. F4 seed was
successfully harvested from the F3 mother plants when they were allowed to regenerate. Fourteen
of the most promising lines were transferred to Tanveer Khan (chickpea breeder, DAWA) as F4
seed from single plants for further variety development or for use as parents in new crosses. A
further bulk of F4 seed has also been transferred to Dr Khan, which will be screened for
ascochyta resistance before being advanced. As few as four seeds were harvested from some of
the susceptible plants since more branches had to be removed for feeding the larvae in the
bioassay. The maintenance of susceptible lines is important if the method is to be used when
screening populations for mapping in the future.
An alternative method developed at CSIRO to screen cotton for budworm resistance and
described by Fitt et al. (1984) was also examined in our preliminary studies to find a reliable
method for screening chickpea. This method is based on feeding larvae excised leaves in a Petri
dish. Leaves are kept moist on filter paper and leaves are replaced as required. Larvae are
weighted after a set period. When we attempted this method the larvae consumed the filter paper
in many of the replicates and we could not be sure if larval weight gain was due to consumption
of chickpea leaves only or due to consumption of cellulose in the filter paper. This problem, and
the question of whether the larvae avoided eating leaves from resistant plants by surviving on the
filter paper, was not resolved.
Conclusions
Screening of chickpea for host plant resistance to budworm was possible using the techniques
described above for measuring antibiosis in larvae by feeding branch cuttings maintained in a
nutrient solution. Correlation was observed between partial resistance in the field and resistance in
cuttings of parental genotypes. Screening could be carried out at any time of year, with more than
one generation per year if required. Since the whole plant was not destroyed, it was possible to
recover seed of the next generation. The effect of feeding larvae on a cutting compared to a whole
plant deserves further investigation. Stress response in the cutting due to excision could interact with
a response to grazing by budworm.
Segregating material from the cross between partially resistant and susceptible was screened at F3,
and those better than the partially resistant parent were selected for further development. True
resistance to budworm was not observed in any plant.
We recommend that the technique should be investigated further and compared to field screening for
a wide range of chickpea genotypes. We anticipate that segregating material, selected from this
screening, should be screened in the field for resistance to budworm, either in Australia or India. The
method should also be tested as a procedure to screen wild Cicer relatives of chickpea.
More recently, Dr James Ridsdill-Smith (GRDC funded project CSE180) has improved the Petridish method of Fitt et al. (1984) further by keeping chickpea leaves fresh in nutrient agar rather
than filter paper. This approach has also given good correlation to field results for wild Cicer
relatives.
Reference
Fitt, g. P., Mares, C. L., & Llewellyn, D. J. (1994). Field evaluation and potential ecological
impact of transgenic cottons (Gossypium hirsutum) in Australia. Biocontrol Science and
technology, 4, 535-548.