Determination of Insecticidal Activity of Kenyan Bt Isolates Against the Spotted Stem Borer, Chilo Partellus
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Determination of insecticidal activity of Kenyan Bt isolates against the spotted stem borer, Chilo partellus
Kimani1,2 G, Nyambaka2 H, Gichuki1, S, Amata1, R, M. Okomo1 and Kasina1 M
1
Kenya Agricultural Research Institute, NARL. P.O. Box 14733-00800 Nairobi
2
Chemistry Department, Kenyatta University,
Correspondence: kasina.j@gmail.com
2
Abstract
The delta – endotoxin crystals containing insecticidal Cry proteins were isolated from 20 unidentified local Bacillus thuringiensis Berliner (Bt) isolates and a reference isolate Bt aizawai (Bta) using froth floatation and low speed centrifugation at KARI Biotechnology laboratories, NARL. The total protein was quantified using the Bradford assay method and protein yield from the nutrient broths was estimated at 3.11 mg/ml ±0.938 mg/ml of nutrient broth culture with a purity level of 54.8% ±15.3% of the protein pellet. Leaf – dip bioassay was used to determine the efficacy of the isolates against Chilo Partellus (Swinhoe), a wellestablished and invasive stem borer in Kenya. Among the isolates, Bt 44 and Bt 48 had the most potent endotoxin crystals to 1st instar C. partellus larvae. In addition, the efficacy of these two isolates was not statistically different (P>0.05) from that of Bta based on LT50 values. The findings show that these two locally available unidentified Bt isolates could be used in management of C. partellus and their characterisation (Cry protein) could aid in their utilization. Key words: Bt efficacy; Bt isolates; LT50; LC50;
Introduction
Maize (Zea mays L.) is the most important staple food crop in Kenya as well as in the subSahara Africa. However, farmers are yet to attain the potential yield per unit area due to a myriad of problems, major ones being infestation and damage by insect pests. Lepidopteran stem borers are generally considered as the most important group of insect pests, causing estimated yield loss of 13.5% annually in Kenya (De Groot, 2002), translating to 400,000 tonnes of maize, equivalent to the amount imported annually. The spotted stem borer (Chilo partellus Swinhoe) is the most prevalent and serious maize stem borers among the five major species reported in Kenya, particularly in low and mid altitude lands (Songa et al., 2002).
These areas are also the most prone to food insecurity. The management of stem borers has
3 largely been based on chemicals, which are rarely effective particularly due to misuse and resistance development by the pest. In addition, most small-scale farmers, who form the bulk of the maize producers in Kenya, cannot afford them (Bonhof et al., 2001). Pesticide misuse impacts on environmental and human health, as well as the non targets that are important in natural pest population regulation. Management of this pest calls for an Integrated Pest
Management systems approach (Camilla, 2000), using friendly practices such as biocontrols.
Microbial pesticides based on Bacillus thuringiensis (Bt) have been used efficiently to control wide range of insect pests (Burges, 1982; Dulmage, 1993).
However, most of the
commercially available Bt products have their strains from temperate zones and their success in the tropics has not been as expected. This has necessitated search for effective local isolates that possess useful attributes such as greater field persistence at high temperatures
(Brownbridge, 1991; Brownbridge and Onyango, 1992; Wang’ondu, 2001; Wamaitha, 2006).
In Kenya, there exists a good number of uncharacterised Bt isolates kept in germplasms at
ICIPE and KARI. The KARI isolates have not been screened against lepidopterans. This study was done to determine effectiveness of some KARI Bt isolate(s) against C. partellus, to form a basis for characterising those effective in terms of the Cry protein content.
Materials and Methods
Study description, isolate activation and multiplication
The KARI isolates used were from the Bt isolate germplasm stored in glycerine. The study was laboratory-based, at KARI Biotechnology Bioassay Laboratory from May to December
2009. Twenty Bt isolates (no. 3, 5, 8, 12, 16, 19, 20, 24, 31, 41, 44, 45, 47, 48, 51, 58, 60, 66,
70, 74) were randomly selected from a pool of 68 Bt isolates and a reference standard Bt aizawai (Bta) isolate also included in the assessment. All isolates were cultured on agar plates prepared by methods described by Poinar and Thomas (1978) and manufacturer’s instructions
(Nutrient agar, Oxoid, Hampshire, England). The petridishes (diameter = 8.5cm) with
4 inoculated agar were incubated for seven days at 30 0C for development of the Bt cultures.
Sporulated cultures were used to inoculate sterile 50 ml liquid broth (Nutrient broth, Biotec
Laboratories Ltd Ipswich, United Kingdom) contained in a 250 ml fluted Elrenmeyer flasks and then incubated on a rotary shaker for 96 h at 30 0C at 200 rpm. At the end of the incubation period, the δ-endotoxin crystals were harvested by shaking the spent culture until frothing occurred. The suspension was allowed to settle for 30 minutes, decanted and centrifuged 4000 rpm for 10 min at 4 0C using a refrigerated centrifuge (Eppendorf 5810C,
Eppendorf-Nether-Hinz, Hamburg, Germany). The supernatant was discarded and the crystals that settled at the bottom of falcon tube to form a pellet were washed thrice by centrifugation in sterile 0.85% saline, air-dried, weighed, suspended in 5-ml saline and stored at -20 0C. The purity of the crystals was ascertained by phase contrast microscopy.
The total protein
concentration of the crystals was ascertained using Bradford-protein determination method using a UV-Vis spectrophotometer (SmartSpec Plus, Bio-Rad laboratories Inc. USA). The
Bradford reagent was prepared by dissolving 10 mg coomassie brilliant blue (G-250) in 5 ml
95% ethanol, adding 10 ml 85% (w/v) phosphoric acid, diluting to 100 ml and filtering the solution through Whatman no.1 just before use. Bovine Serum Albumin (BSA, fraction V) was used as a standard at a stock solution concentration of 2 mg/ml. into appropriately labelled test tubes, 0.1 ml of each standard and unknown Bt samples was pippetted, 5.0 ml of the Bradford reagent added, mixed well and incubated at room temperature for five minutes.
Absorbance was measured at 595 nm for dilutions ranging from 0.1 to 2 mg/ml of standard, a calibration curve of the standard protein was obtained and used to determine the total protein concentration of Bt isolates. The percentage total protein in the pellets of the Bt isolates was also calculated.
Determination of the median lethal concentration of the standard, Bt aizawai, against C. partellus. 5
This was done to identify the right concentration treatment to use for screening the KARI Bt isolates. Five concentrations (13.5 mg/ml, 1.35 mg/ml, 0.135 mg/ml, 0.0135 mg/ml and
0.00135 mg/ml) of Bta were prepared to infect the 1st instar (neonate) larvae of C. partellus at room temperature (27 0C ±1 0C) and 12L: 12D photoperiod. Eggs of C. partellus in the blackhead stage were sourced from KARI – Katumani insectary while natural diet for the larvae (maize leaves) was obtained from two leaved CML 216 maize plants (highly susceptible to stem borers) grown in the KARI biosafety greenhouse level II. Maize leaves (3 cm each) were excised and immersed in 50 ml Bta suspension of the concentration for 3 min in a petridish until all the surfaces of the leaf were covered and then allowed to dry. A single larva was introduced on the petridish and observation made on its feeding. After three days of exposure to the one dose of the toxin, the larva was provided with freshly uninoculated leaves.
A filter paper was placed at the bottom of the petridish and moistened to simulate natural habitat of the pest.
For each concentration, 10 larvae were used each on independent
petridish. Two control treatments were prepared: A leaf with no Bta and another with no leaf.
Larval mortalities were recorded every 24 h. The mean lethal concentration causing 50% larval mortality (LC50) was determined by transforming percentage larval mortalities to probits and plotting these against log transformed concentration values then reading off the concentration corresponding to the probit = 5 using probit analysis of SPSS v13.0.
Bioassay procedure for the KARI isolates
Chilo partellus larvae were subject to a treatment of one dose of a predetermined amount of
Bt toxin. Maize leaves (3 cm each) were excised and immersed in 50 ml Bt suspension of
0.011 mg/ml concentration for 3 min in a petridish until all the surfaces of the leaf were covered and then allowed to dry. The maize leaves were replaced with fresh uninoculated ones after three days in order to subject the larvae to one dose of the treatment. A filter paper was placed at the bottom of the petridish and moistened. Ten 1st instar C. partellus larvae
6 were used per isolate but each into independent petridish. A control without Bt toxin was also included in the assay. Larval mortalities were recorded every 24 h for 144 h and the median lethal time taken for 50% larval mortality (LT50) determined by transforming percentage larval mortalities to probits and plotting these against log transformed time values. The LT50 was determined after 72 h of monitoring and used to rank the Bt isolates in order of increasing effective lethal time which suggests reducing toxicity of endotoxin to C. partellus neonate larvae. Data analysis
The data collected included masses of delta-endotoxin pellets, UV absorbances, concentrations and percentages of total protein in δ-endotoxin pellets and suspensions, mortalities of 1st instar C. partellus larvae exposed to various δ-endotoxins dosages which were subjected to probit analysis to determine LC50 and LT50 values. Where the mortalities of the control were between 5% and 10%, the larval mortalities were corrected using Abott formula ( mortality =
T −C x100%) where T = actual mortality and C = mortality in control)
C
before analysis while those >10% were excluded.
Results
During activation and multiplication of the isolates, the growth of the local Bt isolates and Bta was fast producing smooth creamish-white colonies which were rough edged and slightly raised from the nutrient agar, except for Bt 20 which showed yellowish fluffy growth, probably an indication of contamination of the source. In the nutrient broth, the Bt grew with slight foaming to form a pale yellow opaque suspension that thickened with time and which settled to reveal a white sediment. However, isolate Bt 20 formed a fast settling gelatinous yellow suspension and was excluded from further analysis as it was behaving differently from others confirming indication of germplasm contamination. Under the culture conditions described, lysis of the Bt cells was complete and most of the released protein crystals settled
7 at the bottom of the flask. Almost complete separation of the endotoxin protein crystals from the spores and cell debris was achieved by decantation of the frothy spent culture and low speed centrifugation at 4000 rpm where the crystals formed a white pellet at the bottom of the tube leaving the spores and cell debris in the supernatant fraction. Serial washing, decanting and centrifugation rid the crystals of spent culture components, spores and cell debris.
Microscopy revealed a high concentration of the crystalline inclusions in the pellets obtained.
The mass of the resulting pellets ranged from 0.489 g for Bt 16 to 0.225 g for Bt 47 (mean =
0.314 g ±0.084 g) (Table 1). The protein mass of the pellets was significantly different among the isolates (t16.616, 19, 0.000). Percentage total protein content in the pellets ranged from 26.5% to 92.1% (mean = 54.8% ±15.3%) (Table 1). There was significant difference (t16.014, 19, 0.000) on the percent protein content in the pellets. Isolates Bt 31 and Bt 47 recorded higher contents while Bt 5, Bt 16 and Bt 8 low protein content. The protein yield in the nutrient broth ranged from 2.223 mg to 4.603 mg per ml of broth (mean = 3.112 mg ±0.938 mg) and was significantly different (t23.328, 19, 0.000) across the different Bt isolates.
Table 1: Masses of pellets of Bt isolates and their protein quantities
Protein yield per ml broth (mg)
3.066
3.426
2.350
3.316
3.295
4.282
2.926
4.603
3.324
3.718
2.223
2.770
3.121
3.468
4.042
3.456
3.642
2.223
3.227
2.888
3.263
8
SD
0.084
15.31
0.627
The neonate larvae started feeding immediately after introduction into the petridish and preferred the under side of the leaf. Their feeding intensity slowed down with time and some larvae moved away from the meal after 24 h. Most larvae in the treatment especially with higher δ-endotoxin concentrations stopped feeding after 48 h, appeared weak and stunted in growth compared to the control upon when death was also observed. For instance, with the
13.5 mg/ml endotoxin treatment, 60% of the larvae were found to be away from the diet after
24 h. After 48 h only 10% of larvae were feeding, 40% were found away from the leaves,
20% were weak and 40% of the larvae were dead while all the larvae in the control were actively feeding. Leaf damage was observed to be less on inoculated leaf disks compared to that on the control. Upon death, the larvae appeared dark and shrunk compared to the control.
A dead larva was washed, ground and aseptically inoculated onto nutrient agar plate, creamish growth was observed around the larva which confirmed that the larval mortality was due to ingestion of Bt endotoxins. In the set of starved larvae, mortality was 100% in 48 h.
A 10% larval mortality on the control treatment was observed after 72 h while 30% was recorded after 144 h (Table 2). The cause of this mortality was probably due to drastic changes of weather and laboratory conditions from source of larvae to the bioassay laboratory resulting in weak neonates, dehydration or infection; in the subsequent bioassays, eggs were sourced in the yellow state and allowed to acclimatise to the bioassay lab conditions before hatching, maize leaves were also thoroughly washed with distilled water, the filter paper in the petridish wetted daily with distilled water and the larvae placed back on the leaf if they had moved far away and got trapped under the filter paper which greatly reduced control larvae mortality. The LC50 value estimate for reference isolate Bta was 0.011 mg/ml after 72h
(regression coefficient = 0.31005, 95% confidence limit, SE = 0.13747).
9
Table 2. Percent mortality of neonate C. partellus larvae on treatment with different concentrations of Bt aizawai endotoxins and the LT50 values of each concentration treatment. Toxin
Mean Larvae mortality (%) after time (h)
LT50 (h) concentration 0 h 24 h 48 h 72 h 96 h 120 h 144 h mg/ml 0
0
0
0
10
10
20
30
13.5
0
0
40
90
100
100
100
51.4
1.35
0
0
50
70
90
100
100
53.6
0.135
0
0
10
50
70
100
100
73.1
0.0135
0
0
20
60
60
90
100
70.7
0.00135
0
0
20
40
40
60
60
105.0
Among the different Bt treatments, it is only Bta and Bt 48 that recorded mortality (of 10%) at
24 h of observation (Table 3). However, while Bta increased mortality towards the end of the observation, Bt 48 stabilized at 50% from 72 h. Although Bt 44 recorded the first mortality of
40% at 48 h, it is the only isolate that recorded 100% mortality in the observation period, at
120 h. The Bta recorded 100% mortality at 96 h. No mortality was observed with Bt 51 and the control throughout the observation period. Calculations of LT50 shows that Bt 44 was the most toxic, causing 50% mortality after only 56 h, followed Bta at 64 h and Bt 48 at 73 h.
One-way ANOVA (repeated measures) revealed that the difference in percentage larval mortalities of the standard isolate Bta and the Bt isolates was statistically significant (p0.05) and Bt 44 (F(1.0,5.0) = 0.122, p>0.05).
Table 3. Percent cumulative mortality of neonate C. partellus larvae exposed endotoxins from Bt isolates and the LT50 values of each isolate
Percentage mortality after time (h)
Treatment
0 h 24 h 48 h 72 h 96 h
120 h 144 h
44
0
0
40
70
80
100
100
Bta
0
10
30
60
100
100
100
48
0
10
30
50
50
50
50
24
0
0
10
30
40
70
90
31
0
0
10
30
30
50
60
60
0
0
10
20
30
50
70
66
0
0
10
10
30
30
90
70
0
0
10
10
30
30
30
41
0
0
10
10
10
20
20
16
0
0
0
10
10
10
10
45
0
0
0
10
10
10
10
to 0.011 mg/ml
LT50
at
72 h
56.3
63.5
73.1
90.8
90.8
121.7
314.4
314.4
314.4
567.0
567.0
10
12
8
19
47
74
58
5
3
51
CONTROL
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
20
20
20
10
10
10
0
0
0
0
40
20
20
40
30
10
30
20
0
0
40
20
20
70
60
10
40
40
0
0
-
Discussion
The growth of the Kenyan Bt isolates on nutrient agar and nutrient broth was similar to that reported by other researchers (Brownbridge, 1991; Brownbridge and Onyango, 1992;
Wang’ondu, 2001; Wamaitha, 2006) which illustrates the viability of these isolates at KARI germplasm. However, the growth of Bt 20 isolate showed deviation from the rest probably due to contamination or infection of the stored germplasm.
The protein purity values
recorded by this study were highly variable probably due to the large diversity of isolates that may have differences in optimum growth conditions and variety of insecticidal crystal protein produced (Aronson et. al., 1995) which suggests that the culturing and extraction method may need to be optimised for each isolate in order to obtain equally high δ-endotoxin yields of high purity. The procedure used by this study has been reported to be well suited for largescale production of endotoxin extracts for pesticidal application (Osir and Vundla, 1999). The mean total protein yield from the pellets varied from 20 – 30% similar to what is reported by
Lereclus et al. (1993) although the protocol used was different. Higher protein yield values may have resulted from using lower centrifugation speeds and prior decantation of froth containing cell debris and spores effectively resulting in a lighter pellet.
Two commonly used Bt strains in commercial formulations for control of lepidopterans are Bt kurstaki and Bt aizawai, with the latter strain showing better larval control in situations where
Bt kurstaki is becoming less effective due to resistance development of the pests like the
11 diamond black moth (Schnepf et al., 1998; Polanczyk et al., 2000). The findings of this study closely resemble those of Wang’ondu (2001) with the more toxic isolates (Bt 44 and Bt 48) being obtained from the lowest LT50 values. Significant correlations between endotoxin yield and toxicity among the different isolates illustrates that different Bt isolates produce different δ-endotoxins which may differ in toxicity against different target pests (Uribe et al., 2003).
Recommendations
These results form a basis for further investigation of the local Bt isolates showing significant efficacy against C. patellus such as determination of the Cry proteins therein and how temperature would affect their toxicity. It is also recommended that the toxicity of these isolates be investigated against other local lepidopteran pests in order to determine their target range. Acknowledgement
Authors thank the staff, KARI Biotechnology Centre for support in the laboratory and greenhouse activities. KARI co-funded this study through two projects (Biosafetrain, PI Dr.
R. Amata, and, Characterisation of KARI Bt isolates for management of lepidoterous pests of maize in Kenya, PI Dr. M. Kasina).
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