Efficiency of Bacillus thuringiensis subsp. kurstaki in crude solid fermented matter against the coconut pest, Aceria guerreronis  

Robinson Babysarojam Smitha1 , Prakasan Priji1 , Sreedharan Sajith1 , Niravath Ramani2 , Sailas Benjamin1
1 Enzyme Technology Laboratory, Biotechnology Division, Department of Botany, University of Calicut, Kerala - 673 635, India
2 Acarology Division, Department of Zoology, University of Calicut, Kerala - 673 635, India
Author    Correspondence author
Bt Research, 2015, Vol. 6, No. 2   doi: 10.5376/bt.2015.06.0002
Received: 14 Dec., 2014    Accepted: 23 Feb., 2015    Published: 05 Mar., 2015
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The coconut palm appears to be virtually the only host of coconut mite, Aceria guerreronis Keifer. This serious and exponentially multiplying mite has not succumbed to any control treatments yet, due to its microscopic structure and cryptic life. For the first time, it was explored whether Bacillus thuringiensis subsp. kurstaki (Btk) could be used as an efficient bio-control agent to combat this mite. In a biphasic fermentation strategy for the overproduction of δ-endotoxin, the conventional Luria-Bertani (LB) medium was supplemented with 10% (w/v) potato flour. After initial submerged fermentation of this modified medium for 12 h, the wet and crude solid pellet (i.e., without free solution) with Btk embedded in it was incubated further up to 48 h under solid-state fermentation for obtaining crude fermented matter. A bio-assay set-up was standardized for A. guerreronis on tender coconut button. The probit value (lethal dose) of solid-fermented matter (obtained from potato flour supplemented LB) was 1.64 µg/cm2 with 18 folds more efficiency than the LB control. Preliminary field trial showed that one time application of crude solid-fermented matter in the leaf pockets of the infested coconut palms - prior to monsoon - could be efficient to combat the mite permanently, if all palms in a plantation receive a simultaneous treatment.

Aceria guerreronis; Bacillus thuringiensis; mite, solid-state fermentation; δ-endotoxin; pest biocontrol

The coconut palm (Cocos nucifera L.) (Arecales: Arecaceae) - commonly referred to as “Tree of Life” as well as “Kalpa Vriksha” - provides livelihood to millions of people across the world (Persley 1992; Smith 1998). Globally, coconut occupies an area of 12 million hectares with a total production of about 56 billion nuts per annum. India, Indonesia, the Philippines and Sri Lanka are major coconut-growing countries; they together contribute over 78% of the total world production (Persley, 1992). This crop is attacked by various pests, of which, Dynastes tityus (rhinoceros beetle), Rhynchophorus ferrugineus (red palm weevil), Opisina arenosella (leaf-eating caterpillar), etc. are important. Of late, incidence of coconut nut infesting eriophyid mite, the microscopic Aceria guerreronis Keifer (Acari: Eriophyidae) bearing the vernacular (Malayalam) name Mandari has become a critical problem across the world (Levin and Mammooty, 2003; Lawson-Balagbo et al., 2008). Studies on the geographic pattern of morphological variations among populations of this coconut mite from different countries and continents would provide some bio-geographic information, and it can be concluded that A. guerreronis is of American origin, which was introduced to Asia from Africa, or from the same source as that of the African populations (Navia et al., 2006).
A. guerreronis has a history as a pest of coconut since three decades as of now. The mite, since its detection and description from the Guerrero state of Mexico has been known to invade several countries in the world. In coconut, initial colonization by A. guerreronis requires only one gravid female, the arrival on the button of which is sufficient to establish population to induce further infection to other nuts of the inflorescence, and even a plantation in a short time (Moore et al., 1987). Once the mite has invaded an inflorescence of a coconut, it can spread to adjacent ones through continuous migration by walking. Rapid spread from plantation to plantation and coconut belts are mainly performed through wind, especially during dry season (Haq, 2001). This mite has established the status of a serious pest of coconut in Kerala and other States of peninsular India and neighboring islands (Haq et al., 2002). Rapid invasion, colonization and extensive damage caused by this mite in the coconut belts of India have created serious concerns across the society. Accordingly, much effort has been made to control this pest.
Being microscopic in size and with cryptic habitat, A. guerreronis can reach places that are small enough to be inaccessible to their predators, and they find partial refuge beneath the perianth (cap on the top) of the developing coconut buttons (Haq, 2001). However, some predators can penetrate beneath the perianth of the coconut fruits, and attack the coconut mite. The predatory mites phytoseiidae family, like Neoseiulus baraki (Athias-Henriot) and N. paspalivorus (De Leon) were found prey on A. guerreronis (Reis et al., 2008; Aratchige et al., 2007). Hirsutella thompsonii Pat. (Hypocreales: Ophiocordycipitaceae), a fungus was also found to have some entomopathogenic effects on A. guerreronis (Fernando et al., 2007). Nevertheless, no effective solution is reported yet to combat A. guerreronis attacking coconut palms. Strains of Bacillus thuringiensis (Bt)(Zhou et al., 2014) have been proven as the most applicable biological agent in combating a wide number of insect pests (Fadeland Sabour 2002). However, application of any strain or subspecies of Bt to combat A. guerreronis has not been reported. Under these circumstances, this study addresses the following questions: (a) whether Bt subspecies kurstaki is efficient to successfully combat A. guerreronis; (b) if the answer is ‘yes’, how this biopesticide would be applied to the coconut palm; and (c) how this biopesticide would be produced at a cheaper rate affordable to the farmers in countries where this tree is grown predominantly.
Handling of A. guerreronis is highly risky as it is much sensitive to moisture and exposure to air. In addition to this, since these mites are microscopic and healthy ones are actively moving on the meristematic region of the button within the glass ring, it was very difficult to manage them for counting and photographing. Due to their high sensitivity to moisture, the feed was administered as fine powder with no adhesives. Compared to pellet from LB control, the starchy substrate (due to potato flour) in the potato flour supplemented sample helped the fine powder stick firmly to the buttons, even in the absence of moisture.
Collection of coconut buttons
The coconut palm was affected by A. guerreronis with tender nuts showing different stages of infestation with characteristics injuries i.e., triangular creamy white V-marks on the tender nuts (Fig. 1). One month old coconut buttons were collected to culture the mites at the laboratory conditions, providing suitable temperature and humidity (Fig. 2). The average area of the buttons (perianth region) used in this study was about 8.6 cm2 and diameter 3.23 cm; because A. guerreronis only harbors on this meristematic region (protected by the perianth), sucking the juice.



Figure 1 Habit of Aceria guerreronis (mandari) infested coconut palm with tender nuts showing different stages of infestation. A: A. guerreronis infested coconut palm; B: young button (7 weeks old) showing characteristic V-shaped (triangular) creamy white patch upon infestation; C: 9 weeks old button with multiple V-shaped patches, and D: severely infested button (11 weeks old) shows longitudinal cracks.




Figure 2 Young coconut button and glass rings used for culturing A. guerreronis. A: a 30 days old button with perianth region exposed; B: glass rings used for the culture of mites; C: glass rings attached to the button and sealed with parafilm, and D: culture set- up (with 4 buttons) in a tray.


Life cycle of mites
The life cycle of this mite includes egg, two larval instars and an adult stage, which span about 9-10 days (Fig. 3). The longevity of adult stage was between 3 to 5 days. The pale colored and worm-like elongated adults with two pairs of legs on anterior part of the body are microscopic, measure 200-250 µm in length and 35-50 µm in width. Both nymphs and adults cause injuries on the nut. Each female can lay approximately 30-50 eggs, which are shiny, white and globular in shape. It hatches into larvae (protonymph) in three days. The second instar larva (the so-called nymph) subsequently moults into adults. The populations beneath the perianth of a developing coconut fruit build up rapidly, often producing thousands of mites in each of several aggregations on the same fruit. Massive populations of coconut mites may be present beneath the tepals, the individual parts (tepal) of perianth and on the fruit surface beneath the perianth for about six months during the development of the nut (it takes about one year for a nut to mature), after which the populations decline.



Figure 3 Life cycle of A. gurreronis. A. female mite with eggs; B. protonymph (larva); C. nymph (second install larva); D. young mites; and E.  mature mites.


Before subjecting to bioassay, for acclimatization, many consecutive generations of the mites were made in the laboratory, i.e. in the artificial feeding set up (Fig. 2). The feed used for bioassay was depicted in Figure 4. The standardized mites (a mixed population with specific counts for adult mites) were used for the bioassay. A population of about 20 mature mites with all successive generations was transferred per cm2 on the buttons. Each culture set-up for bioassay was monitored up to 10 days, i.e., until the end of their normal life. It was seen that only mites in the control set-up were passed through all succeeding stages for next generation. About 23% of the mature mites survived after 24 h treatment with 1.25 μg/cm2 crude Btk-toxin (potato flour supplemented), with no intermediate stages (Fig. 5). In all other treatments, all mites (with all stages) were dead within 24 h of treatment (Fig. 5).  It clearly shows that the crude Btk toxin is not only lethal to the nymphal stages, but the adults as well.



Figure 4 Btk toxin used for treatment. A. crude pellet (48 h) of solid-fermented matter containing a mixture of Btk spore, crystal, debris of cells and potato flour (SEM view), i.e., LB was supplemented with 10% (w/v) of potato flour and incubated in a shaker (Scigenics Biotech, India) at 125 rpm and 37 oC for 12 h; the resultant semi-solid viscous fermented matter with embedded Btk was centrifuged (1000 ×g for 10 min), and the  wet solid pellet with no free solution was further incubated (37 oC, static); and B. crude pellet of Btk containing a mixture of spore obtained after submerged fermentation in LB (72 h), vegetative cells, spores, crystal and cell debris are seen (SEM view).




Figure 5 Bioassay for Btk with A. gurreronis. Images were taken at 48 h of treatment.  Various concentrations (1.25μg, 1.88 μg, 2.5 μg, 3.13 μg and 3.73 μg, all per cm2 area) of Btk toxin (dry and powdered crude fermented matter from potato flour supplemented medium, harvested at 48h incubation) was carefully dusted on the tender region culture set-up without disturbing the mites (after initial 24 h observation for their vitality). A is the image of control mites fed with pellet from LB control, and B-F are that of treated mites fed with pellet from potato flour supplemented medium): A. image of mites treated with autoclaved control of potato flour supplemented medium (3.73 μg/cm2) showing most of them are alive and active (above 90%) with both nymphal stages (two adult mites at inset); B. mites treated with 1.25 μg/cm2 fermented matter, here some mites (about 61%) were alive but less active (1 active with 2 dead mites at inset); C. mites treated with 1.88 μg/cm2 fermented matter, here all mites were dead (3 dead mites at inset); D. mites treated with 2.5 μg/cm2 fermented matter, here also all mites were dead (2 dead mites at inset); E. mites treated with 3.13 μg/cm2 fermented matter, all mites were dead (5 dead mites at inset); F. mites treated with 3.73 μg/cm2 fermented matter, all mites were dead (3 dead mites at inset). From this, it is clear that application of pellet from potato flour supplemented medium did not favour the healthy life of the mites with gravid and nymphal stages, which even affected the adult mites.


Probit analysis
Figures 6A and B give a vivid picture of probit analyses. Figure 6A is the probit for the dried and powdered pellet obtained form 72 h old Btk culture in LB (control), i.e., after subtracting the effect of its autoclaved control. At 5 % significance level, the probit value (a concentration required to kill 50% of the target organism) of pellet from LB control was 2.026 µg/cm2, which was 19% less effective than the corresponding treatment of potato flour supplemented pellet (whose probit was 1.639 µg/cm2). For the treatment, only unpurified crude pellet was used. It is advantageous that no further purification of the crystals in the crude fermented matter was required.



Figure 6 Probit analysis. A. Probit graph showing LC50 value of δ-endotoxin (LB control). Mortality at 48 h of treatment was taken for the probit analysis. The probit was 2.026 µg/cm2. B. Probit graph showing LC50 value of δ-endotoxin (pellet from potato flour supplemented medium). Mortality at 48 h of treatment was taken for the probit analysis. The probit was 1.639 µg/cm2.


It has to be noted that the dry weight of pellet obtained from LB control (6.32 mg/mL) was different from that of potato flour supplemented medium (91.25 mg/mLeqv), i.e., the weight of 10% starchy potato flour added to LB must have enhanced the dry weight. 
However, the weight of crude powder in both cases, and their respective autoclaved controls applied per cm2 area on the culture set-up was the same, i.e., 1.25 μg, 1.88 μg, 2.5 μg, 3.13 μg and 3.73 μg (all per cm2 area) concentrations. Hence, the actual toxicity effect (per mL equivalent) of potato flour supplemented pellet is 14.44 folds more than that of LB. If the 19% higher efficacy of flour supplemented pellet, as described in the preceding paragraph is added to this, the total efficacy would be over 18 folds more than LB.
Field trial
The application of crude pellet from potato flour supplemented medium on the field was also found highly effective (Fig. 7).The fermented matter was applied only once on a palm, i.e., prior to monsoon. In one year, the palms which received the treatment produced healthy nuts with almost no attack of the mites.  However, for the successful eradication of the mites, all coconut palms in an area need to be treated simultaneously.



Figure 7 Outcome of limited field trial. A. Coconut palm before treatment with almost no fruits Coconut palm with uninfested maturing nuts (after 6 months of treatment); and C coconut palm with almost mature coconuts (after 12 month of treatment).


We for the first time clearly demonstrated that Bt can efficiently be used for controlling mites (Jisha et al., 2014). Prompted by our previous studies (Smitha et al., 2013a, 2013b), the focus of this work was to check whether the Btk-toxin (crude mixture of a few vegetative cells, endospores, crystals, debris) is suitable to combat A. guerreronis, the highly damaging coconut pest. One might think that A. guerreronis could be controlled by spraying water on the crown of the infested palms (or naturally by rain). Such methods will not be effective because, in such environment, they will be hiding safely within the perianth of nuts (characteristic cryptic habitat) where the reach of water is not possible. Thus, soon after rainy season, their rapid multiplication occurs. This trickiness of the mite attracted us to adopt a suitable strategy, i.e., to apply crude solid-fermented matter within the leaf pockets of the coconut palm at the onset of the rainy season; because the leaf pockets harbor traces of litter and dust, which would act as the solid matter for the growth of Btk, as in the potato flour supplemented preparation, that has already been demonstrated (Smitha et al., 2013a). The copious number of endospores in the solid-fermented matter already deposited in the leaf pockets would germinate during the raining environment, which would facilitate the rapid spread of motile Btk all over the crown of coconut palm. Eventually, Btk would also reach at the hide-outs of the cryptic A. guerreronis thereby killing them, once the toxin is released upon the death of Btk cells. The rapid spread of these mites from one palm to the other is mainly through air currents during dry season and also by crawling or by phoresy (e.g., carried on insects or birds that visit palm flowers) (Haq et al., 2001).
Literature describes no consistent mode of application of Bt-toxins against insects, yet Bt products have been in commercial use for over 50 years. Toxins of Btk have widely been used to control the forest pests such as the gypsy moth, spruce bud worm, the pine procesionary moth, the European pine shoot moth and the nun moth (Gui-ming et al., 2001).  Direct feeding of crude pellet containing Bt-toxin (Fadeland Sabour, 2002), sprays (Mulligan et al., 1980), in the form of pollen diet (Buchholz et al., 2006), application of fermented broths (Brar et al., 2006) are the normal modes of applications being practiced in toxicity assays against various insects. But, none of these techniques seem to be suitable to combat A. guerreronis, because these mites are microscopic, air-borne and highly sensitive to moisture. Moreover, they reside inaccessibly inside the perianth (cap) of the coconut buttons. Thus, the efficacy of starchy fermented matter (powdered) was employed, which would naturally stick to the surface of the coconut buttons proving suitable environment for feeding (ingestion upon sucking the juice from the tender part of the button), avoiding the requirement of an adhesive as in normal applications. This technique is the first of its kind and was found effective for combating A. guerreronis.
Upon application of Btk-toxin, the mortality rates of various insects have been demonstrated by various authors (Aranda et al., 1996; Heckel et al., 1999). We demonstrated a lower probit value (1.639 µg/cm2) for the crude Btk toxin in solid-fermented matter, which indicates the high efficiency of the preparation. Treatment of 0.02 ha pasture plots with Bt H14 (1 kg/ha) resulted in effective control of Aedes spp. and Culex tarsalis. An aerial application on 12 ha duck club pond with Bt H 14 (1 kg/ha) resulted in a 99% reduction of C. tarsalis, apparently without adverse effect on predator populations (Mulligan et al., 1980). Crystals purified from Bt HD1 and HD73 were found highly toxic to tobacco hornworm larvae (50% lethal dose, 0.01 μg per third instars larvae) and crystals purified from Bt HD1 were toxic to meal worm larvae (50% lethal dose 4 μg per second instar larvae) (Zhu  et al., 1989). In these examples, Bt-toxins acted on the actively feeding larvae of susceptible species by a mechanism which involved consumption and proteolytic processing of the toxic protein followed by binding to, and lysis of midgut epithelial cells (Grove et al., 2001). Unlike in the above and many other studies (Broderick et al., 2009) - where larvae were the primary target - in the present study, 100% mortality of the adult mites was noticed in 24 h of application at a concentration of 1.88 μg/cm2 of crude matter, i.e., for a heterogeneous population led by 20 adult mites. Furthermore, data clearly indicate that the adult mites were sensitive to the Btk-toxin.
As seen in the present report, in comparison to the LB control, Btk produced mostly large crystals during SSF.  Until this report, no report is available regarding the efficacy of Bt against the coconut mite, A. guerreronis. From the probit, the dried and powdered form of fermented crude matter with a concentration of 1.88 μg/cm2 was found 100% efficient to control the mites. 
Materials and Methods
Bt strain
The standard culture, Bacillus thuringiensis subspecies kurstaki (Btk) was procured from the Institute of Microbial Technology, Chandigarh, India (strain: BA 83B; MTCC No. 868) and maintained in the conventional Luria-Bertani (LB) medium.
Inoculum and media
Media and crystal production protocols were already published by us (Smitha et al., 2013a; Smitha et al., 2013b). Briefly, two types of media were used for the production of crude toxin. The first one was the standard LB liquid medium (used as control), and the second one was the LB supplemented with 10% (w/v) potato tuber flour; this proportion was standardized, as we already reported (Smitha et al., 2013a, b). Prior to inoculation, both media were autoclaved (at 121 oC for 20 min). The seed culture (12 h) contained approximately 6.5 x 107 colony forming units per mL. Five µl of this seed culture was used as inoculum for every 1 mL LB medium or for every 1 mL LB used to make 10% potato flour supplemented LB. Both media were incubated in an environmental (temperature and humidity controlled) shaker (Scigenics Biotech, India) at 125 rpm and 37 oC, i.e., submerged fermentation (SmF). After initial 12 h fermentation, cultivation strategy was modified for potato flour supplemented medium as already described (Smitha et al., 2013a).
For making the potato flour to supplement LB, the scaly outer rind of locally available mature potato tuber was removed, cut into pieces and dried well in an oven (60 oC for 48 h), and ground into flour using a mixer-grinder. Analytical- and bacteriological-grade chemicals from Chromous (India), Genei (India), Himedia (India), Merck India Ltd., Qualigens (India) and Sigma-Aldrich (USA) were used for the study.
Solid-state fermentation (SSF)
In order to increase the production of δ-endotoxin, after 12 h initial incubation of potato flour supplemented medium, the resultant semi-solid viscous fermented matter with embedded Btk cells was centrifuged (1000 × g for 10 min) to obtain wet-solid pellet with no free solution. The resultant supernatant was used for harvesting extracellular enzymes (Smitha et al., 2013b),  and the solid-pellet was collected aseptically and incubated further up to 72 h (37 oC) to harvest crude Btk toxin (i.e., mixture of endospore and δ-endotoxin). The δ-endotoxin yield was monitored at 12 h intervals. However, LB control was incubated continuously in the shaker up to 72 h.
Selection of palms for collecting coconut buttons
Coconut palms of about 10 m height growing near Calicut University Campus (Calicut University Botanical Garden, Villunnial and Kakkanchery (geographic coordinates: 11°9’N; 75°53’E) at 45-50 M altitude were selected for the present study. Collections were made at about 10 AM in summer (April-May) days with an average day temperature ~33 oC. It is during this time that maximum infestation of the mite is observed in Kerala, the State in India where this work was accomplished.
Collection of coconut buttons
The healthy coconut buttons of about 1 month old from un-infested palms were collected every day and used afresh for the culturing of A.  guerreronis. Hundreds of coconut buttons were used for culturing the mites every week, and the culture conditions were standardized prior to bioassay.
Culturing of A. guerreronis
Mites were collected from infested nuts (Fig. 1). Commercially available glass tubes (borosil) of 5 cm diameter were bored and cut as rings with internal diameter of 2.5, 3, 3.5, and 4 cm with a height of 1.5 to 2.5 cm and 0.5 to 1 cm thickness (Fig. 2). Cover glasses of 1 mm thickness were suitably cut, and used as lids for these culture rings. Coconut buttons (developing small nuts) of about 1 month old were selected from non-infested healthy palms and the appropriate glass ring was fixed with the help of paraffin wax to each button in such a way that the ring touched the boundary between meristematic yellow (the exposed region after the removal of the perianth) and non-meristematic lower greenish region of the nut.  The area (meristematic region within glass ring) of the coconut buttons was calculated using vernier calipers.
About fifty mites (per cm2 area on the bud within the culture ring) were carefully introduced in the meristematic region bordered by the glass ring using a sterile brush. After introducing the mites, the mouth (rim) of the glass ring was lined with a drop of water so that the cover glass on it remains intact. This arrangement considerably helped for observing the behavior of individual stages of the mite through the cover glass and their manipulation according to the need. The culture arrangement was placed in the centre of a plastic tray (8.5 cm diameter and 3. 5 cm height). The tray was then filled with sterile double distilled water up to the lower boundary of the glass ring, so as to prevent the movement of the mites away from the meristematic zone and also to maintain proper humidity. The maximum aseptic conditions were maintained throughout the experiments. Cultures were maintained at 37 oC in an incubator.
Btk toxin for bioassay
Four types of samples were prepared for bioassay. Dried crude pellets obtained from LB control (72 h) and potato flour supplemented media (48 h) were used for treatment. Apart from these samples, their respective autoclaved controls were also used for accuracy in calculation (Fig. 4).
Bioassay for A. guerreronis
For acaricidal assay, the standardized A. guerreronis cultures were used. Bioassay was performed in the culture set-up (24 h old) with about 20 active and mature mites (with all other stages of development) per cm2. Using a fine brush, fine powder (1.25, 1.88, 2.5, 3.13, or 3.73 μg/cm2) of the preparation as above was carefully dusted on to the tender region of the bud within the glass ring. Mortality rate in each treatment was separately scored for probit analyses.
Monitoring the growth and mortality of mites
The life cycle of the mite (Fig. 3) and mortality rates were analyzed by observing through a Magnus compound microscope. The photographs were taken using a digital camera attached to the microscope (Webcam companion 2.0 MEM 1300, Japan).
Field trial
About 5- 10 years old coconut palms in a private property near the University of Calicut campus (11°7′N, 75°53′25″E) seriously infested with A. gureronis were used for the field trial. At the onset of South-West Monsoon (June), 10 g crude solid-fermented obtained from potato flour fermented medium (48 h) was deposited manually in the adaxial side of each leaf base (leaf pocket) of all exposed leaves with or without a bunch of nuts or inflorescence. About 15 such deposits were made per each coconut palm.
Statistical analysis
Lethal concentration LC50 value of Btk formulation against A. guerreronis was estimated from the regression equation of p value from the figure, i.e., p = Intercept + BX (where p is the computer generated table value corresponding to the concentration of the toxin, and X is the calculated value on log10). Antilog of X gives the LC50 dose. Minimum 3 parallel studies were conducted for each datum. SPSS (statistical Package for the Social Sciences) version 20 was used for the probit (p) analyses.
As seen in the probit values, we demonstrated that the crude Btk-toxin in solid-fermented matter was 18 folds more efficient than LB control. It indicates the enhanced production of δ-endotoxin by SSF. The only difference between the two media used was that the latter contained 10% (w/v) potato flour extra. Moreover, the harvest from potato flour supplemented medium (at 48h) was 24 h ahead of the LB control (72h), thus the gestation period for the maximum production of δ-endotoxin has been reduced by 24h. The coconut growing counties predominantly are in the Third World, which look forward for inexpensive methods for combating A. guerreronis. Our strategy shows that one time applications of crude solid fermented matter per palm is sufficient to control the mites permanently, in tune with low cost control measures, as expected by the farmers.
Author’s Contributions
SB designed and prepared the manuscript, RBS did the experiments, PP and SS set the reference and figures, and NR contributed to the culturing of the mite and bioassay.
RBS is grateful to the University of Calicut for granting a Research Fellowship. The support rendered by Sree Chitra Tirunal Institute for Medical Science and Technology, Poojappura, Thiruvananthapuram for obtaining the SEM images is thankfully acknowledged. It is also acknowledged that an Indian Patent application (No. 339/DEL/2012 dated February 7, 2012) has been filed. This manuscript has reference to the PhD thesis of RBS, entitled: “Dual production of endotoxin and amylase from Bacillus thuringiensis subsp. kurstaki by fermentation and efficacy studies of endotoxin against eriophyid mite”. The authors declare that there exists no conflict of interests.
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