Relationship between total carbon, total nitrogen and carbon to nitrogen ratio on growth, sporulation rate and δ-endotoxin synthesis of Bacillus thuringiensis.

The relationship between intracellular and extracellular total carbon, total nitrogen and carbon to nitrogen ratio on Bacillus thuringiensis MPK13 growth, sporulation rate and d-endotoxin synthesis were carried out in shake flask using different types of carbon (glucose, galactose, sucrose, lactose and maltose) and nitrogen (yeast extract) sources. The highest initial intracellular TC (5.15 g/L) and intracellular TN (1.45 g/L) were recorded in medium containing glucose. On the other hand, the highest initial extracellular TC (34.54 g/L) and TN (7.61 g/L) were recorded in medium containing maltose and lactose, respectively. At 48 h of cultivation, the highest final intracellular TC (11.96 g/L) and intracellular TN (3.26 g/L) were also recorded in medium containing glucose. The highest final extracellular TC (27.85 g/L) and TN (7.27 g/L) were recorded in medium containing galactose which showed the lowest growth and sporulation. The presence of d-endotoxin was only detected at 48 h of cultivation using medium containing glucose. The result indicated that high initial intracellular TC and TN values during the cultivation anticipated in high cell growth, sporulation and d-endotoxin production and may be used for indirect measurement of cultivation performance for B. thuringiensis

Identification of Cordyceps javanica and its effectiveness in Controlling Bagworm, Pteroma pendula JOANNIS (LEPIDOPTERA: PSYCHIDAE)

The application of biological control agents as one of the control methods to suppress the infestation of bagworm in Malaysia has developed steadily with the inundation release of formulated entomopathogenic microbes to the outbreak area. In this study, we isolated and identified the entomopathogenic fungi (EPF) from the dead larvae of the bagworm, Pteroma pendula. Mycoses dead bodies of P. pendula were collected from two locations in Kemayan, Pahang, Malaysia. Morphological characterization of EPF was carried out by observing the macroscopic and microscopic growth on Potato Dextrose Agar (PDA) plates using a compound microscope. It was observed that white colonies on PDA changed into purple or pink shades after sporulation. The colony is slow-growing with floccose mycelium, which produces conidiophores with three to four phialides. The conidia were cylindrical to fusiform, smoothwalled and formed in chains on mononematous conidiophores. All the isolates were observed to be similar to Cordyceps fumosorosea features. Molecular identification using universal primer (ITS4 and ITS5) has identified that the isolates were Cordyceps javanica and Parahevansia koratensis. Bioassays of identified isolates were carried out against the second instar larvae of Pteroma pendula showed that C. javanica isolates BSB01 achieved LT50 - 6.76 days and recorded the shortest lethal time compared to the other isolates

Short communication impact of environmental factors on the larval population of bagworm, Metisa plana WALKER (LEPIDOPTERA: PSYCHIDAE) in oil palm smallholdings

Infestations and outbreaks of bagworms, Metisa plana Walker (Lepidoptera:Psychidae) have been reported primarily in Peninsular Malaysia for many years. Bagworm infestation is a significant problem primarily due to the mismanagement and lack of proper monitoring in the field, which later affected the yield and profit loss, especially among smallholders. Understanding the impact of environmental conditions on bagworms may assist in a more proper pest control strategy. Changes in environmental conditions have the potential to interrupt the bagworm's life cycle. This study investigated the effects of temperature, humidity, and rainfall on the bagworm population in selected smallholding oil planted areas in Johor. Anemometer was used to collect the data of temperature and humidity while the collection of rainfall was taken from Meteorology Station. The results showed that temperature (r= 0.211) and rainfall (r= 0.108) have minimal effects on the bagworm population, recording positive relationships with Metisa plana. Relative humidity (r= -0.203), however, showed a negative correlation with M. plana. The findings suggested that temperature and rainfall may affect bagworm populations, but a long-term study is required to comprehend this impact fully. An improve scientific approach should be used in future research along with suitable Integrated Environmental Pest Management (IEPM) techniques, monitoring of the climate and pests, and the use of modelling tools as mitigation strategies to determine the effects of environmental factors in bagworms

Evaluation of artificial diet on growth development of Elaeidobius kamerunicus faust (Coleoptera : Curculionidae)

The rearing of Elaeidobius kamerunicus is difficult as it required the anthesis male inflorescence of oil palm as the breeding and feeding ground for the weevils. To date, utilisation artificial diets for rearing of E. kamerunicus has not been extensively studied. Thus, the objectives of this study were to compare growth development of the larvae using adopted artificial diet formulations and the natural rearing of E. kamerunicus besides determining the nutritional component of the natural food (male inflorescence of oil palm at anthesis). Three artificial diet types adopted from the diet of Anthonomus tenebrosus (Coleoptera: Curculionidae) and Anthonomus grandis (Coleoptera: Curculionidae) were compared with the natural food source towards the growth development of E. kamerunicus. The proximate composition of the natural food source obtained from the field was determined. The findings showed that overall mortality was significantly different for diet types evaluated (P < 0.01). The natural feed caused shorter life cycle of the larvae (10.85±0.34 days). Sex ratio of 0.54 with higher number of female adult was also recorded on natural feed while the artificial diet caused total mortality on the larvae. Nutritional study of the male spikelet at anthesis shown it has 75% moisture content, 20% carbohydrate, 4% protein and less than 1% fat. The formulated artificial diets were found to have lesser essential nutrients to support the growth of larvae. This study provided new knowledge in the formulation of artificial diets and the importance of macronutrient composition on the growth of E. kamerunicus

The insect pest management strategies and their influence on the emergence rate of elaeidobius kamerunicus faust (Coleoptera: Curculionidae) from the post-anthesising male inflorescence of oil palm, Elaeis guineensis

Elaeidobius kamerunicus is the main pollinating insect of oil palm, Elaeis guineensis in Malaysia. Elaiedobius kamerunicus feeds and breeds on the oil palms' male inflorescences. The weevil presence is vital for the successful pollination of oil palm, thus ensuring satisfactory fruit bunch production. MPOB recently reported that the declining quality of oil palm fruit bunch, mainly due to insufficient pollination, had affected the national average oil extraction rate. In part, this scenario was caused by the declining pollinator population. As such, the factors affecting the pollinators' population need to be investigated. One of the critical criteria in estimating the population of E. kamerunicus in the field is to measure the emergence rate of the new adult E. kamerunicus from the post-anthesis male inflorescence (PAMI). The study to investigate the rate of E. kamerunicus adult emergence and their sex ratio was conducted for four months at nine oil palm estates in Malaysia by using PAMI samples that were brought to the laboratory and observed for two weeks. The results have shown that the number of adult weevils that emerged from PAMI was about 36-47 individuals, and the female-to-male ratio for the emerged progenies was 2.33-4.46: 1. Indiscriminate application of broad-spectrum pyrethroid insecticide, cypermethrin has negatively influenced the weevil emergence rate in the field. Whereas, minimal impact on the emergence rate was observed in areas applied with biopesticide. This study showed that the application of cypermethrin could threaten the future population of the pollinating weevil in the field. As such, the excessive application of such insecticides in the field is discouraged and should be replaced by biopesticides to complement other Integrated Pest Management (IPM) strategies. In addition, further investigation is needed to understand how the gender ratio of the progeny could affect the future population of E. kamerunicus

Influence of cultivation conditions on growth, sporulation rate and δ-endotoxin synthesis of Bacillus thuringiensis MPK13

The influence of cultivation conditions on growth of Bt MPK13, sporulation rate,δ- endotoxin synthesis and its toxicity was the focus of this study. The Cry+ strain of Bt MPK13 with consistent ability in δ-endotoxin synthesis was selected by repeated isolation with polymerase chain reaction analysis and δ-endotoxin detection. The effect of monosaccharides (glucose, fructose and galactose) and disaccharides (sucrose, lactose and maltose) on growth of Bt MPK13, sporulation and δ-endotoxin synthesis was carried out using shake flask culture. The data generated was also used to find the relationship between intracellular and extracellular total carbon (TC), total nitrogen (TN) and C/N ratio on growth of Bt MPK13, sporulation and δ-endotoxin synthesis. The effect of dissolved oxygen tension (DOT) control strategies and also the influence of different modes of bioreactor operation (batch, fed-batch and continuous) on the cultivation performance of Bt MPK 13 was investigated using 5 L stirred tank bioreactor. The existence of δ-endotoxin was detected using SDS-PAGE analysis and the toxicity of δ-endotoxin synthesized during different modes of bioreactor operation was carried out against early instars of bagworms, Metisa plana. The selected Cry+ strain of Bt MPK13 was preserved as the stock culture in freeze dried form. The use of Cry+ strain of Bt MPK13 as the stock culture resulted to a consistent 8-endotoxin synthesis in repeated experiments. During cultivation, sucrose, fructose,galactose, lactose and maltose were able to support growth of Bt MPK13, but failed to enhance high percentage of sporulation and the existence of δ-endotoxin was not detected. The synthesis of δ-endotoxin was only detected in cultivation using glucose as the carbon source. Mixture of glucose with other sugars also recorded high cell growth (>1.0 x 1012 cfu/mL) with high percentage of sporulation ( 35%) however, the synthesis of δ-endotoxin was only detected when glucose concentration used in a mixture was higher than 8 g/L, indicating that high glucose concentration (> 8 g/L) must be present in the culture to trigger δ-endotoxin formation.. The highest initial and final intracellular TC and TN were also recorded in cultivation using glucose as the sole carbon source which corresponded well to high cell growth, high percentage of sporulation and δ-endotoxin synthesis. High DOT level (80% saturation) during active growth phase of Bt MPK13 was compulsory for 8-endotoxin synthesis in batch cultivation using glucose (8 g/L) as a carbon source. Enhanced percentage of sporulation (up to 61%) with early 8-endotoxin synthesis (8 h of cultivation) was obtained in cultivation where the DOT was controlled at 80% saturation during active growth and then switched to 60% or 40% saturation at mid-exponential growth phase (after 6 h). The application of this optimal DOT control strategy without affecting the cell growth could also be used to enhance the percentage of sporulation (> 45%) and 8-endotoxin synthesis in fed-batch cultivation. The highest viable cell count (1.5 x 1012 cfu/mL) and spore count (7.1 x 1011 spore/mL) with the existence δ-endotoxin was obtained in fed-batch cultivation with constant feeding of 2 L, 8 g/L glucose at 6 h of cultivation, and optimal DOT control strategy was applied. Compared to batch cultivation, fed-batch without DOT control strategy able to increase cell count (24% increment), however spore count was greatly decreased (50% decrement). Feeding of glucose at stationary phase and intermittent feeding resulted to non-existence of δ-endotoxin. In continuous cultivation, the highest cell (5.8 x 1014 cfu/L.h) and spore (1.6 x 1013 spore/L.h) productivities were obtained at the dilution rate (D) of 0.39 h-1 and 0.05 h-1, respectively. Although the steady-state viable cell concentration (1.7 x 1012 cfu/mL) was substantially higher than those obtained in batch cultivation, more than 50% decrement in spore count was recorded. It is important to note that 8-endotoxin synthesis was not detected in all D tested in continuous cultivat ion,indicating that this cultivation technique is not suitable for Bt production. The culture with the existence of δ-endotoxin recorded 100% mortality towards bagworms (M. plana) at 14 DAT which was related to oil palm defoliation, suggesting that Bt MPK13 has potential to be used as industrial biopesticide. The proposed optimal cultivation conditions may be used in the development of large scale cultivation of Bt MPK13 for subsequent use as biopesticides in oil palm plantation

Effect of different fermentation strategies on Bacillus thuringiensis cultivation and its toxicity towards the bagworm, Metisa plana Walker (Lepidoptera: Psychidae)

The effect of batch and fed-batch fermentation on the cultivation performance of Bacillus thuringiensis was investigated using a 5-l stirred tank bioreactor. Significantly higher viable cell count (> 1.5 × 1012 CFU/ml) was obtained in the fed-batch compared to batch fermentation (1.4 × 1012 CFU/ml). Glucose feeding during the fermentation seemed to enhance cell growth but failed to enhance the sporulation rate. It was found that sporulation and δ-endotoxin synthesis in fed-batch fermentation could be enhanced by the application of optimal dissolved oxygen tension (DOT) control strategy without affecting the cell growth. Fed-batch cultivation with feeding at the exponential growth phase where the DOT was switched from 80 to 40% at 12 h of cultivation recorded the highest spore count of 7.1 × 1011 spore/ml. Cultures obtained from batch cultivation, as well as fed-batch cultivation with feeding at lag or exponential growth phase and the application of optimal DOT control strategy, recorded the presence of δ-endotoxin; however, none was detected in intermittent fed-batch fermentation. Bioassay data against the bagworm Metisa plana Walker (Lepidoptera: Psychidae) recorded the highest corrected mortality (80%) at 7 days of treatment (DAT), using the culture obtained from fed-batch cultivation with feeding during the exponential growth phase, and the DOT was switched from 80 to 40% at 12 h of cultivation. It is important to note that all cultures containing δ-endotoxin exhibited 100% mortality towards M. plana at 14 DAT

Impact of single and two-phase dissolved oxygen tension control on Bacillus thuringiensis cultivation and δ-endotoxin production

Constant δ-endotoxin production is crucial in Bacillus thuringiensis cultivation with aeration strategy playing a significant role in the process. In this study, the impact of DOT control strategies on the cultivation performance of B. thuringiensis was investigated using a 5-L stirred tank bioreactor. Single-phase DOT control recorded low spore count (<5.0 × 1011 spore/mL) and low percentage of sporulation (<45%). Two-stage DOT control cultivation with DOT at 80% during the active growth phase, followed by a switch to 60% or 40% at mid-exponential growth phase contributed to enhanced sporulation (>60%), high maximum cell count (1.5 cfu/mL) with δ-endotoxin formed as early as 8 hr of cultivation. A high maximum specific growth rate, μmax (>0.1 h-1) was also essential in ensuring δ-endotoxin production. High cell count obtained in cultivation with DOT level of 80% saturation during active growth was concurrent with high cell count, high μmax, fast generation time, high OUR, high CER value, and high respiratory quotient (RQ) value. During the two-phase DOT control cultivation, low RQ values were reported. The result showed that the exhaust gas data could be used to monitor B. thuringiensis cultivation performance

Impact of multiple aerial spraying of Bacillus thuringiensis on bagworm control in oil palm smallholdings in Johor, Malaysia

The outbreak of bagworm has been a severe threat, causing significant loss to the oil palm industry in Johor, Malaysia. This study investigated the impact of multiple aerial applications of Bacillus thuringiensis (Bt)-based biopesticides in controlling the bagworm outbreak at two smallholdings in Johor, namely Smallholding A (Sh.A) and Smallholding B (Sh.B). Two types of agricultural aircrafts used in this study were Grumman G-164 A Super AgCat and M-18 Dromader. The results showed that a significant reduction of bagworms (83.5%) was recorded after the third round of aerial spray in Sh.A. Whilst, Sh.B recorded a significant reduction of bagworms (83.5%) after the fourth round of aerial spray. The result indicated that multiple applications of Bt aerial spray at the precise timing and strategy based on the bagworm’s life cycle are crucial in ensuring the application’s effectiveness in bringing down the bagworm population to below the economic threshold level (ETL). A census conducted in 2019 and 2020 recorded that the bagworm population in both areas maintained below the threshold level even after more than three years of application. With the implementation of a long-term Integrated Pest Management (IPM) strategy, such as planting beneficial plants, the bagworm population can be maintained under ETL even after years of aerial sprays

Automatic Classification of Bagworm, <i>Metisa plana</i> (Walker) Instar Stages Using a Transfer Learning-Based Framework

Bagworms, particularly Metisa plana Walker (Lepidoptera: Psychidae), are one of the most destructive leaf-eating pests, especially in oil palm plantations, causing severe defoliation which reduces yield. Due to the delayed control of the bagworm population, it was discovered to be the most widespread oil palm pest in Peninsular Malaysia. Identification and classification of bagworm instar stages are critical for determining the current outbreak and taking appropriate control measures in the infested area. Therefore, this work proposes an automatic classification of bagworm larval instar stage starting from the second (S2) to the fifth (S5) instar stage using a transfer learning-based framework. Five different deep CNN architectures were used i.e., VGG16, ResNet50, ResNet152, DenseNet121 and DenseNet201 to categorize the larval instar stages. All the models were fine-tuned using two different optimizers, i.e., stochastic gradient descent (SGD) with momentum and adaptive moment estimation (Adam). Among the five models used, the DenseNet121 model, which used SGD with momentum (0.9) had the best classification accuracy of 96.18% with a testing time of 0.048 s per sample. Besides, all the instar stages from S2 to S5 can be identified with high value accuracy (94.52–97.57%), precision (89.71–95.87%), sensitivity (87.67–96.65%), specificity (96.51–98.61%) and the F1-score (88.89–96.18%). The presented transfer learning approach yields promising results, demonstrating its ability to classify bagworm instar stages