Evaluation on the Toxicity Effect of Four Bacillus Thuringiensis Strains Against the Teak Skeletoniser, Paliga Damastesalis Walk
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Journal of Tropical Forest Science 12(3): 425-430 EVALUATION ON THE TOXICITY EFFECT OF FOUR BACILLUS THURINGIENSIS STRAINS AGAINST THE TEAK SKELETONISER, PALIGA DAMASTESALIS WALKER (LEPIDOPTERA: PYRALOIDEA: CRAMBIDAE) J. Intachat*, M. Mastura Forest Research Institute Malaysia, Kepong, 51209 Kuala Lumpur, Malaysia & H. Staines Division of Mathematical Sciences, University of Abertay Dundee, Bell Street, Dundee, Scotland DD1 1HG, United Kingdom Received July 1998 INTACHAT, J., MASTURA, M. & STAINES, H. 2000. Evaluation on the toxicity effect of four Bacillus thuringiensis strains agains e teath tk skeletoniser, Paliga damastesalis Walker (Lepidoptera: Pyraloidea: Crambidae). Four Bacillus thuringiensis (Bt) strains, namely HD-1, Florbac, SN-2 (Bt subspecies aizawai strain) and SN-5, were screened against the 3rd instar of the teak skeletoniser, Paliga damastesalis. A total of 750 larvae were tested in the exposur h . Mortalit2 laborator7 °C analyse1 s d + an ewa 5 y8 2 afte 4 t , dya usin24 r g logistic regression t subspecieB . s aizawai (strai ne mos th SN-2 founs e t b effectiv wa )o t d n ei controlling the teak skeletoniser larvae. Key words: Teak skeletoniser - Paliga damastesalis - Tectona grandis - Bt strains - toxicity - Malaysia INTACHAT , MASTURAJ. , STAINES& . M , . 2000H , . Penilaian kesan ketoksikan empat strain Bacillus thuringiensis terhadap perangka daun jati, Paliga damastesalis Walker (Lepidoptera: Pyraloidea: Crambidae). Empat strain Bacillus thuringiensis (Bt), iaitu HD-1, Florbac, SN-2 (strain subspesie t aizawai)sB SN-5n da , diuji terhadap instar ketiga perangka daun jati, Paliga damastesalis. Sebanyak 750 larva diuji di dalam makmal pada suhu . Kematia°C 1 2 5± n selepas pendedaha dianalisim ja 2 7 n ns da menggunakaselam 8 4 , a24 n regresi logistik. Subspesie aizawait sB (strain SN-2) didapati paling berkesan dalam mengawal larva perangka daun jati. Introduction Ever since teaplantes kwa d widel Malaysiyn i earle th yn a i 1990s, leave younf so g teak saplings have been observe constantle b o t d y skeletonise larvae motha th f y o eb d . The moth Paliga damastesalis Walker (Lepidoptera: Pyraloidea: Crambidae), commonly known as the teak skeletoniser, was later found to be different but closely related to e teath k skeletoniser specie Indin i s a that causes similar damag n teaeo k leaves (Intachat 1998). They seem to be present wherever teak is planted in Malaysia, especially in Sabah causing, usually, low levels of defoliation. Attacks by this moth are seasonal (Chey 1996) but can be severe particularly in Sabah during dry spells. *Present address: Owley Farm, South Brent, Devon TQ10 9HN, England, United Kingdom. 425 426 Journal of Tropical Forest Science 12(3): 425 - 430 Wit currene hth t increased interes tean i t k plantin Malaysian gi , which extendo st small-scale plantings populatione th , thif so s mot expectee har increaseo dt . Constant defoliation, especially on young teak saplings, may have adverse effects on their growth. It is therefore important that a suitable, environmentally friendly control be applie proteco dt t young teak plantings from heavy defoliation causemothe th y db . One possible approach is biological control using a microbial pesticide. Preparations of the bacterium Bacillus thuringiensis Berliner (Bt) have been widely used as a microbial pesticide since the 1960s in the United States. Besides exotoxins, Bt also produces a glycoprotein entomotoxins called 5-endotoxins (Singleton & Sainsbury 1987) e nature endotoxinth Th . f o e s differs amon t subspeciegB d an s isolates, wit characteristice hth thesf so e specific endotoxins determining their target organism. Amon e besgth t know d mosnan t widely use t insecticidedB e thosar s e formulate dkurstakin frova t mB isolates tha pathogenie ar t toxid can c onl larvao yt e of butterflies and moths (Lepidoptera). Bt var. aizawai is another Bt that kills caterpillars (Weinzerl & Solter 1995). As a biological control approach is suitable for forest environments, the usage of Bt to control major forest pests from the order Lepidoptera may be an effective solution. However, because Bt strains are specific, we evaluated the toxicity of four different Bt strains in search of one, which might effectively control P. damastesalis. No previous analysis has investigated the effects of various Bt strains on this species of teak skeletoniser. Material methodd san s The micro-organisms used in this study consisted of four strains of Bt: HD-1, Florbac, SN-2 and SN-5. All the strains were obtained from the Department of Microbiology, Faculty of Life Science, Universiti Kebangsaan Malaysia. Strain HD-1 was previously obtained from the Bacillus Genetic Stock Center, The Ohio State University Columbus, U.S.A. Florbac strai re-isolates nwa d fro commerciae mth l preparatio Florbay nb , cFC Novo Nordisk, Denmark. Strain SN-2 is a radiation-resistant mutant strain of a re- isolated strain (subspecies aizawai) from a commercial preparation (Bacillex, Shionogi, Japan) (Jangi & Hashim 1983) and SN-5 strain is a local isolate of Bt subspecies kurstaki. Media were sterilised by autoclaving at 120 °C for 15 min and all subsequent manipulations were carried out in a glass laminar flow cabinet. Liquid transfers were made with sterilised pipette tips. The bacterial strains were grown and maintained nutrienn o t agar slant werd san eunde C store° 4 rt da aerobi c conditions t strainB . s were subculture inoculatiny db gloopfua nutrienf o l l intm 0 ot1 brot incubated han d e resultanTh . °C t2 inoculum± 0 3 t a h s fo8 wer4 r e then adjusted furthe obtaio t r n e turbiditth y comparabl thao et f McFarlano t d Standar 5 (Vandepitt0. d. No al t e 1991) before they were ready to be tested. McFarland No. 0.5 was chosen since this is the lowest bacterial suspension standard that is equivalent to the density of bacteria at 150 x 105 cells per ml (Anonymous 1995). Teak leaves wer t intecu o roun diameterm c d 0 disc tea9. e f so Th .k discs were then washed in a 10% solution of sodium hypochlorite (NaOCl), rinsed in a series of distilled water air-driedd an , . Distilled wate nutrienr ro t broth (both bein controe gth l foue th treatments rf o strain e f bacterion so r o ) a suspension thes swa n sprayen do 7 42 Journal of Tropical Forest Science 0 43 12(3) - 5 :42 both teae sideth kf sdisco s usin ordinarn ga y handspray (Taiwan SC-202) totae Th . l amount of two sprays on each teak disc sprayed by the same person was found to be equivalen . Fivinstad ml e3r 1 r o larvat t describes (a e Intachan di t 1999) obtained from laboratory rearin starved gan threr dfo e hours were then introduce eaco dt f ho the treated teak discs that were kept individually in Petri dishes at 25 ± 2 °C in an incubator. For each of the six treatments, a total of 25 Petri dishes (replicates) were used and hence a total of 125 larvae were tested for each treatment. mortalite Th larvaye ratth f eacn eeo i h replicat eacf eo h treatmen recordes twa d . Mortalith 2 7 d yan rate 8 r som4 afte sfo , e24 r treatment/time combinations were less than 10%, so the normal approximation to the binomial distribution and hence the use of the arc sine transformation was invalid. Stepwise logistical regression was use determino dt relative eth e importanc crossee th f eo d factors exposure timd ean treatment and the nested factor dish (replicate). Only factors that significantly reduced significanc% 5 e th t (a e level e residuath ) l deviance were include e modelth n di . Attempts at model simplification by combining treatment levels were made to determine significant differences among treatments. Standard diagnostic checks showed no departures from the usual model assumptions. Results and discussion Mortalities were observe groupn di larvaf so e treated amon foue gth r strains within . Infecte h firse 4 th 2 t d larvae showe dlosa appetiten si , ceased movin becamd gan e flaccid and brownish in colour. Fluid of disintegrating internal tissues liberated from the broken skin was also observed in dead larvae. Since the 5-endotoxins produced contaca t no e t poisonar t byB larvae th , e must first inges leae th tf disc coated with effective b Bo t tdeatd occuy an e hma r within several hours thereafter. Ingestioy nb susceptible larva will degenerate the mid-gut epithelium and paralyse the gut. This may kill the larvae directly or they may stop feeding and die within 48 to 72 h from e effectth f lethao s l septicaemia (blood poisoning) (Singleto Sainsburn& y 1987, Weinzer Solte& l r 1995). Table 1. Mean mortality (%) for each treatment after 24, 48 and 72 h exposure. The fitted value for each treatment/time combination is shown in parentheses. Time Treatment code Treatment 24h 48h 72h Tl Control 1 2.4 9.6 23.2 (distilled water) (1.76) (7.78) (25.65) T2 Control 2 0.8 7.2 24.0 (nutrient broth) (1.57) (6.97) (23.45) T3 HD-1 1.6 10.4 51.2 (3.85) (15.84) (43.51) 4 T Florbac 4.0 17.6 41.6 (3.85) (15.84) (43.51) T5 SN-2 5.6 20.8 56.0 (5.79) (22.43) (54.19) T6 SN-5 4.8 13.6 26.4 (2.39) (10.34) (32.06) 428 Journal of Tropical Forest Science 12(3): 425 - 430 Observatio showeh 4 2 t ndhighea a r percentag f mortaliteo SN-r SN-d yfo 2an 5 (5.d 4.86an % respectively compares a ) Florbao dt HD-d can 1 (1.4.0d 6an % respectively) (TablslighA .