Efficacy of Bacillus thuringiensis (var. kurstaki) Against Diamondback Moth (Plutella xylostella L.) Eggs and Larvae on Cabbage Under Semi-Controlled Greenhouse Conditions.

The efficacy of Bacillus thuringiensis (var. kurstaki) (Btk) against the diamondback moth (DBM) on cabbage was studied at Botswana College of Agriculture, Gaborone, Botswana. Using five concentrations of Btk: 2, 4, 6, 8, and 10 g/L, bioassays were conducted against DBM eggs and second instar larvae at 30°C ± 5°C. Each treatment was replicated three times. Probit analysis was used to determine the LD50 and LD90 values for the treatments against eggs and larvae. When the treatments were assessed at 72, 96, 120, and 144 hours, LD90 values against larvae were 11.02, 10.22, 5.92, and 4.01 g/L, whereas they were 7.71, 6.94, and 6.24 g/L against eggs when assessed 48, 72, and 96 hours after the expected time of hatching. This indicated that Btk was effective against both eggs and larvae when exposed for long periods. The slopes of the probit lines for larvae assessed at 24, 48, 72, 96, 120, and 144 hours after application were 0.250, 1.064, 0.910, 0.383, 0.453, and 0.414, while those against eggs were 1.153, 1.246, and 0.933 when assessed 48, 72, and 96 hours after the expected time of hatching. This indicates a smaller change in mortality with increase in pesticide dosage for both eggs and larvae. Btk treatments achieved 85.7%-94.6% reduction in DBM damage on cabbage. Therefore, Btk can be used to achieve effective control of DBM eggs and larvae and reduce damage on cabbage under greenhouse conditions.

Introduction

Cabbage (Brassica oleracea var. capitata L.) is an extensively grown vegetable around the world.1 It is among the most popular food crops in Botswana and grows well in many parts of the country.2 However, its production is seriously affected by a wide range of pests including the diamondback moth (Plutella xylostella L.) (DBM), bagrada bug (Bagrada hiliaris Burn), and the cabbage aphid (Brevicoryne brassicae L.).3 The most serious among these is DBM, which has a cosmopolitan distribution; it is believed to be the most universally distributed species among the Lepidoptera, and occurs wherever brassicas are grown.4 DBM was first recorded as an important pest of cabbage in Southern Africa as early as 1917.5 It is highly migratory, and its seasonal movements have been well documented.4 Its exceptional pest status is due to several factors: the diversity and abundance of host plants, the disruption of its natural enemies, its high reproductive potential (with over 20 generations per year in the tropics), and its genetic elasticity, which leads to rapid development of resistance to insecticides.6,7 DBM is most destructive in areas where there is frequent application of insecticides. In Botswana, the control of DBM relies heavily on the use of synthetic insecticides.8 However, it has been demonstrated that DBM quickly develops resistance to many new insecticides.9,10 It has reportedly developed resistance to most synthetic pyrethroids, organophosphates, carbamates, and actinomycetes in many cabbage growing areas of the world;8,11 this represents a serious threat to its effective management. Unfortunately, in Southern Africa, control of DBM is still heavily dependent on these conventional synthetic pesticides.8 Such pesticides can affect nontarget organisms in both treated and untreated fields. Therefore, efforts to promote the use of microbial insecticides as alternatives are continually being made, and one such alternative is the use of Bacillus thuringiensis.

The most successful of the microbial products are those based on Bacillus thuringiensis Berliner (Bt). B. thuringiensis (Bt) var. aizawai and var. kurstaki (Btk) products are recommended for the control of DBM in South Africa.12 Bt is a spore-forming, soil-inhabiting bacterium that produces a selectively toxic protein in the form of a crystal within the cell. This protein inclusion is the active component in Bt products. It consists of a protoxin form of one or more α-endotoxins. When this inclusion is ingested by a susceptible host, it is solubilized and the protoxin is processed to the active δ-endotoxin form. The insecticidal crystal proteins are divided into five major classes, namely cry 1, cry 2, cry 3, cry 4, and cry 5, with specific insecticidal activity against Lepidoptera, Lepidoptera and Diptera, Coleoptera, Diptera and Lepidoptera, and Coleoptera, respectively.13 The active toxin binds to specific receptor sites on the gut epithelium, leading to slow degradation of the gut lining and starvation. Bt slows development, lowers survival, and reduces rates of larval feeding. The toxins also affect the muscular and nervous systems.14 Thus, several days are required to kill insects that have ingested Bt products.14 Death generally occurs within 1–3 days.15 Effective commercial Bt products for the control of caterpillars have been available all over the world for over 20 years and have been used against lepidopteran pests of brassicae crops for many years.13

With the increasing costs and risks posed by synthetic pesticides, advances in biotechnology have facilitated efficient production of microbial insecticides. As a result, the use of Bt in pest control has become an important tool. The use of Bt in most cabbage growing areas of the world is likely to increase in the future. Munthali et al16 suggested that the use of Bt is the most effective method of controlling DBM in areas where resistance has not yet been reported.

Microbial insecticides, particularly those derived from Bt, offer a great deal of promise for pest management. Bt insecticides are highly effective against certain pests, yet they do not harm humans, most beneficial insects, and other nontarget organisms.17 Thus Bt insecticides do not pose the serious environmental hazards associated with conventional pesticides.

Although resistance to conventional insecticides is widespread,18 few cases of resistance to Bt have been reported.17 However, potential for development of pest resistance is a threat to the continued success of Bt.15 Increased use of Bt is likely to intensify selection for resistance in the field. Development of substantial levels of resistance to Bt in DBM has been documented for two field populations from watercress and one from cabbage in Hawaii.17

The usefulness of Bt for pest management has been increased by the development of new strains and recent advances in genetic engineering, including the insertion and expression of Bt toxin genes in major crop plants and in plant-colonizing bacteria.15

Several factors influence the effectiveness of this entomogenous bacterium when it is employed in microbial insect control. During the application of the bacterium, a suspension of minute particles is deposited on the plant foliage. From the time the bacterium leaves the application equipment until it is consumed by the target species, it is exposed to all the fluctuations of the environmental conditions. The period of exposure will vary depending on the feeding habits and activities of the target insect. During this period, sunlight and temperature appear to be major deleterious environmental factors.19 Burges and Hussey19 found that low temperatures in the field would allow more exposure of the bacterium to solar radiation, rainfall, etc, and would reduce the effectiveness of the bacterium. Higher temperatures would allow the bacterium to kill the insect before solar radiation could have its effect.

Because of its low toxicity to many beneficial insects, Bt is suitable for use in integrated pest management (IPM) programs, especially where pests have developed resistance to other insecticides.20 The application of Bt as a component of an IPM program can reduce environmental pollution, deleterious impact on beneficial entomofauna, and delay the expression of resistance to other pesticides.7 However, little is known about its effects when applied against DBM eggs and larvae. This study evaluated the efficacy of Btk on DBM eggs and larvae under semicontrolled greenhouse conditions.

Materials and Methods

Experiments were conducted at the Botswana College of Agriculture, Gaborone, Botswana (24°34′ 25″S, 25°95′0″E; altitude: 998 m) from June to December 2010 in cages that were placed in a greenhouse at an average temperature of 30°C ± 5°C and a day length average of 9.8 h per day. The cabbage seedlings were initially raised in nursery trays and transplanted into small black plastic sleeve pots filled with loam soil; each pot was 12 cm in diameter and 15 cm in depth. Cabbage seedlings at the five-leaf stage were used to rear the DBM to ensure adequate host substrate for oviposition of eggs by adults. The seedlings were watered regularly adlib to prevent wilting. Nine potted plants were placed in each of six insect-rearing cages. Each cage was 45 cm long, 45 cm wide, and 40 cm high, and was covered with clear lumite netting of 32 mesh size to prevent pest infestation from natural populations or escape of insects from the artificially infested plants in the cage. Every cage had a door with a sleeve that was used during watering of plants, artificial infestation of the plants, feeding of the adult insects, the application of sprays, and the removal of plants during pest and plant damage assessments.

Bioassay methods

Btk, Vectobac® (3000 ITU/mg) (soluble granules), registered and locally available for use in Botswana, was used in the bioassay experiment. A small hand-held trigger sprayer that produced a fine spray of a relatively narrow range of droplet sizes was used to apply the spray solutions. Six treatments comprising five Btk concentrations (2, 4, 6, 8, and 10 g/L water) and distilled water were used. The manufacturer’s recommended rate (4 g/L) was included as a check. Each treatment had nine seedlings. The sprays against eggs were applied when each plant had between 50 and 70 eggs, and those against larvae were made when plants had between 30 and 40 larvae each. Each seedling was sprayed separately. The bioassay was repeated three times. This gave a total of 54 plants per bioassay and 162 sprayed plants altogether. Each pot had a label, which indicated the treatment and its date of application.

The bioassay was conducted on eggs and second instar larvae (the first instar larvae are leaf miners, which are not susceptible to a pesticide with a stomach poison mode of action such as Btk). DBM eggs used in the bioassay were obtained by placing 50 laboratory-bred pupae in each of six insect rearing cages that contained nine potted cabbage seedlings. Adults emerging from the pupae were left to oviposit on the seedlings for 4 days before they were removed from the cages. Each seedling was examined using a hand lens at ×10 magnification, and the eggs laid on the leaves were counted. Each artificially infested seedling was sprayed with one of five different insecticide concentrations or distilled water for control.

Assessment of egg and larval mortality

As viable DBM eggs take an average of 4 days to hatch at 25 ± 5°C,21 treatments against eggs were applied 3 days after oviposition. The eggs oviposited on each plant were counted immediately before application of treatments followed by counts at 48, 72, and 96 hours. Egg mortality was determined by comparing the number of viable eggs prior to application of treatments with the numbers found after treatment. The eggs found unhatched after each treatment were considered dead. For larval mortality, eggs were allowed to hatch into first instars and develop into second instar larvae. The first instar larvae are leaf miners and second instar larvae are surface feeders, therefore they were easy to differentiate; these were counted before treatment. The larvae were assessed at intervals of 24, 48, 72, 96, 120, and 144 hours after treatment. Any larvae that did not show signs of life after prodding with a needle were counted as dead.

Plant damage assessment

Plant damage assessments in each treatment were conducted 14 days after the DBM eggs had hatched. The total number of leaves per plant was recorded, the number of leaves with damage symptoms was counted, and the results were used to calculate the percentage of damaged leaves per plant. When the tiny DBM larvae hatch from the eggs, they penetrate the leaf and immediately begin to feed between the upper and lower epidermis, removing the mesophyll tissue and the chlorophyll and leaving the clear upper and lower epidermis intact, which is called a “window”. The number of windows per leaf for each plant was recorded and used to estimate the intensity of damage caused per plant. The experiment was repeated three times. A single assessment was conducted at day 14 of each of the three trials.

Data analysis

Probit analysis22,23 was used to analyze the mortality results. The mortality data were transformed to probits, while the dosages were transformed to log10(x + 1) before analysis. LD50 and LD90 values were estimated from the probit lines. Relative susceptibilities of eggs and second instar larvae were compared using LD50 values and slopes of probit lines. LD90 values were used to compare the mortalities caused by the recommended dosage to those that were achieved by the other dosage levels at different periods of exposure to Btk.

The results on percentage seedling damage were transformed to arcsines before analysis in order to normalize them. Using the MSTATC24 statistical package, analysis of variance (ANOVA) was used to analyze the plant damage data. Averages were separated using the Tukey’s Honestly significant difference test25 where significant effects were found.

Results

DBM egg mortality

Figures 1–3 show a positive linear relationship between log dose and mortality (transformed to probits) caused by Btk on DBM eggs (r2-values of 0.983, 0.956, and 0.999, respectively). Figure 1 indicates that the LD50 and LD90 of Btk against DBM eggs when assessed 48 hours after expected hatching of eggs was 3.79 and 7.71 g/L, respectively. The recommended dose (4.0 g/L) of Btk gave a probit value of 0.52 (equivalent to 46.15% egg mortality) during this period. When the assessment was done at 72 hours after expected hatching period, the LD50 and LD90 values of Btk were 2.60 and 6.94 g/L, respectively (Fig. 2). The mortality caused by the recommended dose was 0.61 on the probit scale (which is equivalent to 51.35% egg mortality). The LD50 and LD90 values of Btk were 1.75 and 6.24 g/L, respectively, 96 hours after they were expected to have hatched (Fig. 3). The recommended dosage achieved 0.81 on the probit scale (equivalent to 64.16% egg mortality).

Figure 1

Probit mortality of DBM eggs exposed to different doses of Btk assessed 48 hours after expected time of hatching.

Figure 2

Probit mortality of DBM eggs exposed to different doses of Btk assessed 72 hours after expected time of hatching.

Figure 3

Probit mortality of DBM eggs exposed to different doses of Btk assessed 96 hours after expected time of hatching.

Table 1 shows that the average mortality of DBM eggs per plant was significantly affected by both Btk concentration and period after application. The interactions were significant (ANOVA, P < 0.05%). The greatest mortality (100%) occurred on plants treated with 8 and 10 g/L dose assessed 48 hours after the eggs were expected to have hatched, while the lowest (24.0%) was on plants treated with 2.0 g/L assessed 48 hours after the expected incubation period of eggs (Tukey P = 0.05) (Table 1). The overall treatment averages show that Btk caused the greatest average egg mortalities (76.8%) when treatments were assessed 96 hours after expected egg incubation period and that the lowest (66.0%) occurred 48 hours after the expected egg incubation period.

Table 1

Effect of Btk concentrations and period of exposure on egg mortality.

PERIOD AFTER EXPECTED DATE OF HATCHING (HOURS)	2 g/L	4 g/L	6 g/L	8 g/L	10 g/L	OVERALL PERIOD AVERAGES
48	24.0g*	46.0de	60.0c	100.0a	100.0a	66.0c**
72	38.0f	51.0d	75.0b	100.0a	100.0a	72.8b
96	44.0ef	64.0c	76.0b	100.0a	100.0a	76.8a
Overall treatment averages	35.3d***	53.7c	70.3b	100.0a	100.0a	71.9

Notes:

Interaction averages in the body of the table followed by the same letters are not significantly different (Tukey’s Honestly significant difference test (P < 0.05).

Averages in the column followed by the same letters are not significantly different (Tukey’s Honestly significant difference test (P < 0.05).

Averages in the row followed by the same letter are not significantly different (Tukey’s Honestly significant difference test (P < 0.05).

DBM larval mortality

Figures 4–9 show a positive linear relationship between the log dose and probit mortality caused by Btk. Figure 4 indicates that Btk insecticide did not achieve 50% larval mortality 24 hours after application at all the concentrations. The recommended dose (4 g/L) achieved 0.074 (equivalent to 15.79%), 0.157 (equivalent to 23.34%), 0.563 (equivalent to 48.62), 0.740 (equivalent to 59.34%), 0.832 (equivalent to 65.80%), and 1.0 (equivalent to 100%) larval mortalities 24, 48, 72, 96, 120, and 144 hours after exposure, respectively. The LD50 values achieved with application of Btk when assessed 48 and 72 hours (Figs. 5 and 6) were 6.71 and 3.07 g/L, respectively. The LD90 values achieved with application of Btk when assessed 72, 96, 120, and 144 hours were 11.02, 10.22, 5.92, and 4.01 g/L, respectively.

Figure 4

Probit mortality of DBM larvae 24 hours after application of different doses of Btk.

Figure 5

Probit mortality of DBM larvae 48 hours after application of different doses of Btk.

Figure 6

Probit mortality of DBM larvae 72 hours after application of different doses of Btk.

Figure 7

Probit mortality of DBM larvae 96 hours after application of different doses of Btk.

Figure 8

Probit mortality of DBM larvae 120 hours after application of different doses of Btk.

Figure 9

Probit mortality of DBM larvae 144 hours after application of different doses of Btk.

Results in Table 2 show that both concentration and period after Btk application significantly affected the average mortality of DBM larvae per plant (ANOVA, P < 0.05%). The interactions were also significant. The greatest mortality (90.8, 92.3, 93.0, and 93.3% per plant) occurred 144 hours after application of 4.0, 6.0, 8.0, and 10.0 g/L Btk solutions, while 91.7% mortality occurred 120 hours after application of 10 g/L. The recommended dose (4 g/L) took 144 hours to achieve 90% larval mortality. The results also show that the least mortality (0.0–26.7% per plant) occurred in the control treatment throughout the study period. Mortalities that occurred in the control treatment 144 hours after application of treatments were similar to those achieved 72 hours after application of 2.0 g/L, 48 hours after application of 4.0 g/L solution, and 24 hours after application of 6.0, 8.0, and 10.0 g/L Btk solution (Tukey, P < 0.05). The overall treatment averages show that Btk concentrations also had a significant effect on the mortality of larvae. Overall larval mortalities differed significantly from each other and increased in the order 44.8 < 53.6 < 61.5 < 63.9 < 68.7% on plants treated with 2.0, 4.0, 6.0, 8.0, and 10.0 g/L, respectively. The overall exposure period results were also significantly different and increased in the order 17.9 < 34.2 < 48.1 < 57.8 < 66.9 < 77.1 when assessment was done 24, 48, 72, 96, 120, and 144 hours after application.

Table 2

Effect of Btk spray concentration and period of exposure on DBM larval mortality.

PERIOD AFTER APPLICATION (HOURS)	CONTROL	2 g/L	4 g/L	6 g/L	8 g/L	10 g/L	OVERALL PERIOD AVERAGES
24	0.0j*	13.3ij	18.4hij	23.3ghij	25.8ghij	26.7fghij	17.9f**
48	0.0j	23.3ghij	26.7fghij	48.3defgh	50.0defg	56.8cdef	34.2e
72	2.2j	40.0efghij	51.7cdefg	60.7bcde	65.1abcde	68.9abcde	48.1d
96	12.2ij	60.0cde	63.4abcde	67.5abcde	69.2abcde	74.7abcd	57.8c
120	16.7ij	65.6abcde	70.3abcde	76.7abcd	80.8abc	91.7a	66.9b
144	26.7fghij	66.7abcde	90.8ab	92.3a	93.0a	93.3a	77.1a
Overall treatment averages	9.6d***	44.8c	53.6bc	61.5ab	63.9a	68.7a	50.4

Notes:

Interaction averages in the body of the table followed by the same letters are not significantly different (Tukey’s Honestly significant difference test (P < 0.05).

Averages in the column followed by the same letters are not significantly different (Tukey’s Honestly significant difference test (P < 0.05).

Averages in the row followed by the same letter are not significantly different (Tukey’s Honestly significant difference test (P < 0.05).

DBM damage on cabbage plants

Table 3 shows the effect of different insecticide concentrations on the intensity of cabbage leaf damage per plant. One-way ANOVA shows that the average damage was significantly (ANOVA, P < 0.05) affected by the insecticide concentrations used. The least damage (14.3%, 12.7%, and 3.3% per plant) was achieved with application of 6, 8, and 10 g/L of Btk, while the greatest damage (81.3%) occurred on untreated plants followed by plants treated with 2 and 4 g/L, respectively (Tukey, P < 0.05).

Table 3

Effect of Btk spray concentrations on intensity of cabbage leaf damage per plant.

	0 g/L	2.0 g/L	4.0 g/L	6.0 g/L	8.0 g/L	10.0 g/L
Treatment averages	81.3a*	46.0b	40.7b	14.3c	12.7c	3.3c

Notes:

Averages in the row followed by the same letter are not significantly different (Tukey’s Honestly significant difference test (P < 0.05).

Discussion

The finding in this study that Btk caused egg mortality is unexpected because other researchers (eg, Gould)26 found that Btk requires to be ingested to be effective. Since eggs cannot acquire the lethal dose of an insecticide like Btk through feeding, it can be deduced from the findings that Btk uses other modes of action to cause mortality of DBM eggs. The results found in the present study, where application of the recommended dose of Btk did not achieve effective control over the 96-hour period, suggests that longer periods are required to achieve high egg mortality. Since the bulk of Btk activity is lost within 2–3 days of application under field conditions,15 effectiveness of Btk for periods longer than 72 hours would be expected to decline. However, applications higher than the recommended dose achieved 100% control 48 hours after the expected period to hatching. Toxicity data with Btk insecticides is limited, but Burges and Hussey19 found that Btk had insecticidal activity on DBM through inhibition of the development of the embryo and that the insecticidal effects against eggs were considerably lower than against larvae. Bond and Boyce27 demonstrated that, when viable DBM eggs on cabbage leaves were dipped in solutions of Btk, mortality increased with increase in Btk concentration. This explains the results found in this study, where egg mortalities increased significantly with increase in Btk concentration.

From the results in Figures 4–9 and Tables 2 and 3, several observations can be made. Longer exposure periods are required to achieve 90%–100% DBM larval mortality; the recommended dose did not achieve 90% larval mortality during the study period, Higher dosages than the recommended dosage are required to effectively protect cabbage plants from DBM damage. It is therefore logical to assume that the longer it takes to kill sufficient numbers of larvae, the greater the damage caused by the larvae on the crop. After ingestion of Btk, the active toxin is known to bind to and destroy the midgut epithelium, resulting in rapid gut paralysis, which causes the larva to stop feeding within hours in the most sensitive species.15 Btk-affected larvae die from starvation, which may take several days. Since Btk does not kill rapidly, users may incorrectly assume that it is ineffective if treatments are assessed a day or two after application. The finding that the use of higher concentrations of Btk achieved 90% larval mortality 120 hours after application shows that much higher concentrations of Btk are required to achieve adequate control of the pest and protect the crop from serious damage in a greenhouse. The results in this study show that, apart from the dosage that should be used against a pest species, information on the label of an insecticide should include periods of exposure required to achieve the required level of control of the target pest. Such information would enable farmers to decide on the periods between spray applications.

Comparisons of slopes of probit lines can also provide an indication of relative toxicities of insecticides.28 An insecticide with a steep slope would therefore be expected to provide faster pest mortality with change in dose. The slopes of the probit lines in Figures 4–9 show that large increases in the dosages are needed to cause significant increases in mortality of DBM larvae when Btk is applied against DBM larvae. The fact that dosages higher than the recommended dosage of Btk took longer than 120 hours to achieve 90%–100% mortality shows that higher concentrations can be used to achieve effective control of DBM provided longer exposure periods are provided. This suggested that the recommended dosage was too low to achieve effective DBM larval mortalities. These results are similar to those found by Talekar,15 who suggested that use of higher doses of Btk than those previously used in control programs might reduce pest populations sufficiently and avoid the need for repeat applications in the following year. Burges and Hussey19 also reported that control efforts with Bt failed because of insufficient doses and differences in potency of the preparations used.

In order to determine the effectiveness of the pesticide in the management of DBM, it is important to consider the level of pest reduction achieved as well as the reduction in crop damage as a result of application of the pesticide. When results in Table 2 are considered together with those in Table 3, it appears that 90.8% larval mortality is required to effectively protect cabbage using Btk. This shows that the level of mortality that resulted in effective protection of cabbage plants required a dosage higher than the recommended dosage of Btk (of 4.0 g/L).

Conclusions and Recommendations

The objective of applying insecticides against crop pests at the recommended dose is to ensure the production of large quantities of high-quality crop yields by using minimum amounts of the active ingredient. It can be concluded from this study that Btk can offer effective control of DBM eggs and larvae and prevent serious damage to cabbage provided long exposure periods are allowed. Higher Btk dosages than recommended by the manufacturer are required to offer effective control of DBM under greenhouse conditions. Since Btk sprays caused a considerable level of mortality of DBM eggs, it can be concluded that apart from requiring ingestion, Btk also uses other unidentified mechanisms of killing target pests. Further research is needed to investigate the mode of action used by Btk sprays against sessile, nonfeeding life stages of DBM. This study also showed that, when applied under high temperature conditions, Btk persisted and caused mortality of eggs for up to 3 days after application. Since this study was conducted under semicontrolled greenhouse conditions, further research is needed under field conditions to validate the results obtained in the present study.