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Rice is an important crop and a staple food in Malaysia. Herbicides are used extensively to control weeds, which represent a major constraint to yield production. Although the introduction of Imidazolinone-resistant Rice with its management system (IRPS) has greatly improved both yields and weed control, the system is designed to be used for only a short term before transitioning to local varieties. Thus, a survey was conducted among 115 farmers to obtain information on their general knowledge on weed control and IRPS. The results showed that the majority of the farmers use herbicides to control all types of weed presented, with a small minority still using manual control. The majority of farmers using IRPS were applying the herbicide imidazolinone when soil condition were right, and only once per season, which is the recommendation. Most of the farmers still utilized imidazolinone to control weedy rice but would not use it on other weeds. However, many of the farmers perceived imidazolinone as becoming more ineffective and expensive and were willing to change to other herbicides if there was a viable alternative. Although herbicide is the main method employed in controlling weeds when using IRPS, farmers still regard imidazolinone as an ineffective herbicide. The reason IRPS is still in use is due to the high yields provided. This study shows a better understanding of knowledge on weeds and IRPS among farmers. Nonetheless, the IRPS will become a redundant system due to the ineffectiveness of imidazolinone and a new system should be introduced to replace it.

Sidi MB, Wasli ME, Polly E, Jaffar ANNM, Kalu M, Sani H, Nahrawi H, Elias H, Omar D. 2021. Short Communication: Incidence of insect pest on planted Shorea macrophylla at reforestation sites in Gunung Apeng National Park, Sarawak, Malaysia. Biodiversitas 22: 5162-5168. Incidence of insect pest in Shorea macrophylla (de Vriese) P.S. Ashton had critical foliage damage in mono planting technique. The main objectives were to assess the foliage damage intensity of planted S. macrophylla by age stands and type of foliage damage. The insect pest attacks the foliage of S. macrophylla was determined. The study site was located at Gunung Apeng National Park (GANP), Sarawak, Malaysia, with planted S. macrophylla in enrichment planting at different years (planted in 2008, 2009, 2010, and 2011 for age stand 6, 5, 4, and 3, respectively). The results showed that the degree of foliage damage decreases with the age stand of S. macrophylla tree. Therefore, foliage damage was suspected to be caused by insect pests. Among the common foliage damages observed was "hole damage" caused by insect order Lepidoptera. Although foliage damage was significant, the severity of the damage will "heal" as the age stand increases. Further investigation on other possible causes of these pest attacks should be initiated to find solutions that may hasten the growth of planted S. macrophylla for forest restoration.

This study evaluates the efficacy of palm oil-based nanoemulsion insecticides in thermal fogging applications against adult Ae. aegypti. The nanoemulsion formulations contained a palm oil methyl ester solvent, water, a non-ionic surfactant, and active ingredient deltamethrin, with nanoemulsion droplet diameters ranging from 362 to 382 nm. Knockdown and mortality rates of caged mosquitoes were measured at various distances up to 18 m from the spray nozzle. After 15 min of insecticide exposure, nanoemulsion insecticides achieved a knockdown rate of >97% at a spraying distance of 4 m, and the knockdown effect increased substantially with exposure time. At an 18 m spraying distance, the best nanoemulsion formulation, NanoEW8, achieved a high mosquito mortality rate of more than 80%, whereas the non-nanoemulsion and the commercial product reached only 14 and 8 m distances, respectively, for comparable mortality. The artificial neural network (ANN) was used to predict the mosquito knockdown distribution over the spraying distances and time intervals. The models predicted that NanoEW8 can still cause knockdown at a maximum distance of 61.5 m from the discharge point 60 min after spraying. The results established that Ae. aegypti was susceptible to the newly developed palm oil-based nanoemulsion insecticide, indicating a high potential for mosquito control.

Simple Summary The use of Metarhizium anisopliae as a biological control of insect pests has been experimented in the laboratory as well as in field trials. This includes against the termite Coptotermes curvignathus, however the results have varying degrees of success. One reason could be due to the lack of detailed knowledge on the molecular pathogenesis of M. anisopliae. In the current study, the conidial suspension of M. anisopliae isolate PR1 was first inoculated on the C. curvignathus, after which the pathogenesis was examined using two different approaches: electron microscopy and protein expression. At the initiation stage, the progression observed and documented including adhesion, germination, and penetration of the fungus on the cuticle within 24 h after inoculation. Later, this was followed by colonization and spreading of the fungus at the cellular level. Proteomics of C. curvignathus witnessed the expression of proteins related to cell regulation and defense, while in M. anisopliae, protein related to transport and fungal virulence were expressed throughout the infection. These findings offer relevant knowledge for use in the development of M. anisopliae as a prospective biological control agent for termites in the future. Abstract Metarhizium anisopliae (Metchnikoff) Sorokin, a pathogenic fungus to insects, infects the subterranean termite, Coptotermes curvignathus Holmgren, a devastating pest of plantation trees in the tropics. Electron microscopy and proteomics were used to investigate the infection and developmental process of M. anisopliae in C. curvignathus. Fungal infection was initiated by germ tube penetration through the host’s cuticle as observed at 6 h post-inoculation (PI), after which it elongated into the host’s integumental tissue. The colonization process continued as seen from dissemination of blastospores in the hemocoel at 96 h PI. At this time point, the emergent mycelia had mummified the host and forty-eight hours later, new conidia were dispersed on the termites’ body surface. Meanwhile, hyphal bodies were observed in abundance in the intercellular space in the host’s body. The proteomes of the pathogen and host were isolated separately using inoculated termite samples withdrawn at each PI-time point and analyzed in two-dimensional electrophoresis (2-DE) gels. Proteins expressed in termites showed evidence of being related to cell regulation and the immune response, while those expressed in M. anisopliae, to transportation and fungal virulence. This study provides new information on the interaction between termites and its entomopathogen, with potential utilization for developing future biopesticide to control the termite population.

Water hyacinth (Eichhornia crassipes) is a free-floating aquatic plant and is also widely cultivated as an aquatic ornamental plant in Malaysia. In June 2018, a severe foliar disease with typical leaf blight symptoms were observed on leaves of water hyacinth plants (approximately 50%) in waterways adjacent to two rice fields located at Tanjung Karang and Sungai Besar, Selangor province, Malaysia. Symptoms appeared irregular necrotic lesions with concentric rings, later lesions expanded to entire leaves and became blighted. Twenty symptomatic leaves were collected from two sampling locations. Symptomatic leaf tissue was cut into small pieces (5 × 5 mm), surface sterilized with 0.5% sodium hypochlorite (NaOCl) for 2 min, rinsed three times with sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 25 °C with a 12-h light/dark cycle for 7 days. Twenty single-spore isolates were recovered from sampled leaves, all isolates exhibited Paramyrothecium-like morphology and two representative isolates, PR1 and PR2 were used for further studies. Fungal colonies were initially white aerial mycelia with sporodochia bearing olivaceous green conidial masses formed on PDA after 5 days of incubation. Conidiogenous cells were phialidic, hyaline, smooth, straight to slightly curved, 13 to 20 × 1.0 to 1.8 μm and setae were absent. Conidia were aseptate, hyaline to pale green, smooth, cylindrical to ellipsoidal with rounded ends, and measured 5.8 to 8.0 μm × 1.8 to 2.2 μm (n=50). These morphological characteristics were consistent with the description of Paramyrothecium roridum (Tode) L. Lombard & Crous (Lombard et al. 2016). Total genomic DNA of the isolates was extracted from fresh mycelium using DNeasy Plant Mini kit (Qiagen, USA). The internal transcribed spacer (ITS) and calmodulin (cmdA) gene regions were amplified using the ITS5/ITS4 (White et al.1990) and CAL-228F/CAL2Rd primer sets (Carbone and Kohn 1999; Groenewald et al., 2013), respectively. BLASTn analysis showed that the ITS and cmdA sequences of the isolates were 100% identity with Paramyrothecium roridum ex-epitype strain CBS 357.89 (GenBank accession nos. KU846300 and KU846270), respectively. The resulting sequences were deposited in GenBank (ITS: Accession nos. MW850370, MW850371; cmdA Accession nos. MW854363, MW854364). Pathogenicity tests of the two isolates were performed by spray inoculation on healthy leaves of each five potted water hyacinth plants using a 3-ml conidial suspension (1 × 106 conidia/ml) produced on 7-day-old PDA cultures incubated at 25 °C with a 12-h light/dark cycle. Five potted water hyacinth plants inoculated with sterile water served as controls. Inoculated plants were covered with plastic bags for 48 h to maintain high humidity and kept in a growth chamber for 2 weeks at 25 ± 1°C, 95% relative humidity and a 12-h light/dark period. The experiment was repeated twice. Eight days post-inoculation, symptoms on inoculated leaves developed necrotic brown lesions similar to those observed in the field, while control leaves remained asymptomatic. After 2 weeks of inoculation, lesions enlarged into severe blighting until all leaves died. Paramyrothecium roridum was re-isolated from randomly selected symptomatic tissues and verified by morphology and sequencing of ITS (MZ675387, MZ706462) and cmdA (MZ686706, MZ712041) loci, confirming Koch's postulates. The fungus was not re-isolated from non-inoculated control plants. Pa. roridum is distributed on a wide range of plants (Farr and Rossman 2021) and has been reported to cause leaf spot of water hyacinth in Nigeria (Okunowo et al. 2013) and Sri Lanka (Adikaram and Yakandawala 2020). To our knowledge, this is the first report of Pa. roridum causing leaf blight of water hyacinth in Malaysia. This disease is an emerging threat to water hyacinth and it reduces the leaf quality, therefore, appropriate management should be developed to control this disease.

The tobacco whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a cryptic species complex with members capable of inducing huge economic losses. Precise identification of members of this complex proves essential in managing existing populations and preventing new incursions. Despite records of serious outbreaks of this pest in Malaysia little is known about species status of B. tabaci in this region. To address this, a comprehensive sampling of B. tabaci from different host plants was conducted in 10 states of Malaysia from 2010 to 2012. Members of the complex were identified by sequencing partial mitochondrial cytochrome oxidase subunit I (mtCOI) gene and constructing a Bayesian phylogenetic tree. Seven putative species were identified including Asia I, Mediterranean (MED), China 1, China 2, Asia II 6, Asia II 7, and Asia II 10. The most important finding of the study is the identification of the invasive MED species from locations without previous records of this species. All putative species except Asia I and MED are recorded from Malaysia for the first time. This study provided the first introductory map of B. tabaci species composition in Malaysia and emphasizes the urgent need for further studies to assess the status of MED invasion in this country.

The current study aimed to investigate the chemical composition and insecticidal activity of Artemisia herba-alba essential oil obtained by hydrodistillation and solvent extraction against papaya mealybug Paracoccus marginatus. The essential oil was analysed using gas chromatography-mass spectrometry and the insecticidal activity was recorded for different times and concentrations under laboratory conditions. According to GC-MS results, the chemical components detected of essential oil isolated by ‎hydrodistillation and solvent extraction were 52 and 62 compounds of both extraction methods, the main components of essential oil of hydrodistillation method were eucalyptol (32.81%), &alpha-pinene (28.62%) and linalol (10.18%) while the major components of essential oil isolated by solvent extract were pentadecanoic acid (16.69%) and dihomo-‎‎.gamma.-linolenic acid (15.2%). The essential oil of hydrodistillation method exhibited more insecticidal ‎activity than solvent extract method against P. marginatus, 1000 mg/L of essential oil isolated by ‎hydrodistillation achieved 100% ‎mortality of papaya mealybugs after 120, ‎‎168 h of treatment with LC50 values which ‎was‎‎ 65.362 mg/L after 168 h from treatment and LT50 value which was ‎22.386 h‎‏‎‎ at 1000 mg/L.‎ The findings reveal and propose that the essential oil of A. herba-albais a rich by bioactive components and it is a prospective source of botanical insecticides.

This report describes the development of environmentally-benign nanoemulsion formulations for control of adult Sitophilus oryzae(L., 1763) (Coleoptera: Curculionidae) and Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae). The formulations were emulsions of neem [Azadirachta indicaA. Juss (Sapindales: Meliaceae)] oil, nonionic surfactant (alkylpolyglucoside or polysorbate 80), and water using low-energy emulsification method. Four formulations were chosen and physicochemical characterization showed a particle size range of 208-507 nm in diameter. Bioassays were conducted using food and filter paper impregnation methods. The formulation comprised of polysorbate surfactant with 2.0 mL/kg azadirachtin caused a 100% mortality of S. oryzaeadults after only 24 h of exposure, with the food impregnation method. The toxicological studies carried out at Putra University of Malaysia toxicology laboratory between November 2014 and December 2015 indicated that S. oryzaeadults were more susceptible than T. castaneumadults to nanoemulsion formulations including 2.0 mL/kg azadirachtin according to median lethal times., Bu çalışmada, Sitophilus oryzae(L., 1763) (Coleoptera: Curculionidae) ve Tribolium castaneum(Herbst, 1797) (Coleoptera: Tenebrionidae) erginlerin mücadelesinde kullanmak için çevre dostu nano emülsiyon formülasyonlarının geliştirilmesini amaçlanmıştır. Emülsiyon formülasyonu, düşük enerjili emülsiyonlaşma yöntemi kullanılarak neem yağından [Azadirachta indicaA. Juss (Sapindales: Meliacear)], iyonik olmayan yüzey aktif maddeden (alkilpoliglukosid veya polisorbat 80) ve sudan oluşturulmuştur. Dört formülasyon seçilmiş ve fizikokimyasal çapı 208-507 nm olan partikül boyutu aralığı ile karakterize edilmiştir. Biyoassay çalışmaları, besine ve filtre kağıdına emdirme yöntemleri kullanılarak yürütülmüştür. 2.0 mL/kg azadirachtin konsantrasyonlu polisorbat yüzey aktif maddeden oluşan formülasyon besine emdirme yöntemi ile sadece 24 saat maruz bırakıldığında S. oryzaeerginlerinin %100 ölümü gerçekleşmiştir. Kasım 2014 ile Aralık 2015 tarihleri arasında Putra Malezya Üniversitesi toksikoloji laboratuvarında yürütülen toksikolojik çalışmalar ortalama ölüm zaman değerlerine göre S. oryzaeerginleri 2.0 mL/kg neem yağı içeren nano-emülsiyon formülasyona karşı en hassas tür olduğunu göstermiştir.

The experiment was conducted at the Toxicology laboratory, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Malaysia in 2013. Allelopathic potential of aqueous methanol and water extracts of M. cochinchinensis leaves, seed and root was investigated on seed germination and seedling growth of goosegrass and biotest crop species: lettuce (Lactuca sativa). The treatments consisted of five concentrations (100, 75, 50, 25, 0 %); plant parts (leaves, seed, root) and extraction solvents (methanol, water) were replicated three times and arranged as a completely randomized block (CRD) design. Germination, radicle and hypocotyl growth of all test plant species were inhibited at concentrations (100, 75, 50 and 25%). The total germination percentage was lowest with the methanolic extracts of leaf, seed and root of M. cochinchinensis at 100 % concentration in the order of 0.00, 30.66 and 4.66%, respectively. Concomitantly, the radicle length inhibition percentages of methanolic extracts at higher concentration of 100% were 100, 88.5 and 94.4% of the leaf, seed and root extracts, respectively. The water extracts recorded the highest germination percentage and lower inhibitory activity of the radicle and hypocotyl length. The study confirmed plant growth-inhibitory compounds of M. cochinchinensis is dependent on the extraction solvents and extract concentrations as expressed that methanolic solvent at higher extract concentration had the stronger inhibitory activity.

Parasitic Echinochloa colona (L.) Link has globally invaded paddy fields that could devote to severe grain yield loss. Prolonged extensive application of commercial synthetic herbicides could pose the weed evolved resistance, bioaccumulation and environmental deterioration. Hence, this paper pertains to the aim of investigating potent natural products which have shown considerable phytotoxic activities, subsequent phytotoxin studies and extraction optimization for pre-emergent control of E. colona. In the phytotoxicity study, a suite of invasive plants was procured and the leaf, stem and root extracts were evaluated by in vitro germination and seedling growth assays. The invasive plant extracts exhibited multiple magnitudes of inhibition against E. colona in the plant species-, plant part- and concentration-dependent manners. Along with the dose-response study, the leaf extracts of Mikania micrantha Kunth ex H.B.K., Clidemia hirta (L.) D. Don , Dicranopteris linearis (Burm.f.) Underw. and Ageratum conyzoides L. showed the attainable half maximal effective doses (ED50) below the concentration 100 g biomass dry weight equivalent (BDWE)/L, indicating the top-ranked inhibition of E. colona among the investigated plant extracts. The top-ranked phytotoxic leaf extracts were subjected to qualitative and quantitative characterizations including extraction yield, total phenolic content, phytochemical screening and spectroscopic analysis of the phytochemicals with possible inhibitory effect. Joint putative allelochemicals consisting of 16 phenolics and 5 aromatics were detected concurrently by liquid chromatography-mass spectrometry (LC-MS), were found contributed to the appreciable germination inhibition. Amongst the abundant phenolic acids were protocatechuic acid, gallic acid, p-hydroxyphenylacetic acid and chlorogenic acid. To suit the industrial desire in product development, the phytotoxic leaves were processed through extraction optimization using response surface methodology (RSM). A multivariate face-centered cube design (FCCD) was established to seek the functional relationship between the process variables (extraction time, agitation speed and solvent consumption) and the response (germination inhibition). Quadratic response surface models were obtained with the models significance and significant effect of the process factors at 95% confidence interval were examined by various statistical analyses. Verification experiments at the optimal conditions were well matched with the predicted values, along with absolute errors in the range 3.63% to 6.70%, indicating the proposed quadratic models were valid and useful prediction of the extraction conditions. These outcomes provide the valuable findings on the possible use of potent invasive plants as bioeffective, economical and eco-friendly herbicide alternative towards fostering the sustainable weed management.

This study aimed to improve the efficacy of azadirachtin (Azadirachta indica. A. Juss) against two serious pest species of stored products, Sitophilus oryzae (L.) and Tribolium castaneum (Herbst), through nano-emulsion formulations. Pseudoternary phase diagrams were constructed consisting of an emulsion system of an active ingredient (neem oil), surfactant (polysorbate or alkylpolyglucoside), and water. Isotropic regions were formed in the pseudoternary phase diagrams, and four formulations were selected from the isotropic regions and characterized according to particle size, particle aging, zeta potential, stability and thermostability, surface tension, viscosity, and pH. The selected formulations showed particle sizes of 208-507 nm in diameter. The result of contact toxicity demonstrated excellent mortality of S. oryzae and T. castaneum adults, with a mortality range of 85-100% and 74-100%, respectively, at a 1% azadirachtin concentration after only 2 days of exposure. Compared to non-formulated neem oil, the nano-emulsion formulations significantly increased the mortality of the tested species.

Bacterial leaf blight (BLB) and sheath brown rot (SBR), caused by Xanthomonas oryzae pv. oryzae (Xoo) and Pseudomonas fuscovaginae, respectively, are bacterial diseases that lead to substantial yield losses in rice. Natural plant-based products represent a sustainable alternative to combat bacterial diseases due to their biodegradability and overall safety. However efficient ways of delivering them are crucial to their success. In an attempt to maximize the antibacterial properties of botanical bactericides for the control of these pathogens, this study evaluated the efficiency of different emulsion formulations of Piper sarmentosum extracts. The emulsion formulations were demonstrated to be effective in controlling BLB and SBR of rice in in vitro plate assays and in planta under glasshouse conditions. The observed in vitro inhibition of the bacterial pathogens and significant disease suppression in planta indicate that these plant extract formulations represent promising alternatives to be adopted in management strategies for controlling rice diseases., Agricultural science; Microbiology; Plant biology; Natural product chemistry; Bacterial leaf blight; Biological control; Sheath brown rot; Piper sarmentosum; Rice; Plant-based formulations.

Influence of waterborne butachlor (BUC), a commonly used pesticide, on morphometric, biochemical, and molecular biomarkers was evaluated in juvenile, full sibling, diploid and triploid African catfish (Clarias gariepinus). Fish were exposed for 21 days to one of three concentrations of BUC [mean measured µg/L: 22, 44 or 60]. Unexposed (control) triploids were heavier and longer and had higher visceral-somatic index (VSI) than diploids. Also, they had lighter liver weight (HSI) and showed lower transcript levels of brain gonadotropin-releasing hormone (GnRH), aromatase (cyp191b) and fushi tarazu-factor (ftz-f1), and plasma testosterone levels than diploids. Butachlor treatments had no effects, in either diploid or triploid fish, on VSI, HSI, weight or length changes, condition factor (CF), levels of plasma testosterone, 17-β estradiol (E2), cortisol, cholesterol, or mRNA levels of brain tryptophan hydroxylase (tph2), forkhead box L2 (foxl2), and 11 β-hydroxysteroid dehydrogenase type 2 (11β-hsd2). Expressions of cyp191b and ftz-f1 in triploids were upregulated by the two highest concentrations of BUC. In diploid fish, however, exposures to all BUC concentrations decreased GnRH transcription and the medium BUC concentration decreased ftz-f1 transcription. Substantial differences between ploidies in basal biomarker responses are consistent with the reported impaired reproductive axis in triploid C. gariepinus. Furthermore, the present study showed the low impact of short term exposure to BUC on reproductive axis in C. gariepinus.

Pot experiment was conducted under glasshouse conditions to investigate the effects of the inoculation of N2 fixing Stenotrophomonas maltophilia strain Sb16 and application of three herbicides (paraquat, pretilachlor and 2,4-D) at 0, 1/2X, X and 2X their recommended field application rates (X) on endophytic bacterial populations and physio-morphological parameters of aerobic rice. The physio-morphological traits such as plant height, leaf area, chlorophyll content, nitrogen (N) content, root dry mass, root length, root volume and root average diameter were assessed at 60th day after the treatment. Data on endophytic bacterial counts were collected at 15, 30, 45 and 60 days after the treatment. Results obtained from the study revealed that the number of endophytic bacteria and physio-morphological characters of aerobic rice significantly decreased with increasing herbicides dose. Sb16 inoculation significantly (P ≤ 0.0001) increased all the parameters measured. N contents were the highest (2.53 %) in the inoculated samples treated with half dose of 2, 4-D; but the lowest contents (1.89 %) were obtained in the non-inoculated samples treated with double dose of paraquat. The results suggest that Sb16 strain can improve productivity of aerobic rice under herbicide-stressed soil.

SUMMARYRice productivity is limited by many pests, especially Pomacea spp. in Southeast Asia. Pomacea spp. damage to rice depends on sowing methods, flooded conditions, and snail densities in the field. Therefore, this study aims to evaluate the effect of different sowing methods, water levels, and snail density (1, 2, and 3 snails per plot) on the damage potential of Pomacea maculata and Pomacea canaliculata to rice and its yield. Both species caused complete loss of crop in direct seeding and 14 days old transplanted rice. The least damage by both species was recorded in 21 and 28 days old transplanted rice with no further damage after week five. Irrigation and snail density also influenced damage whereby highest damage was recorded in rice grown with 5 cm water level in comparison to 2 cm. At 2 cm water level, damage by various snail densities was trivial. However, in 5 cm water level, damage increased with the increasing snail density and the highest damage was observed at three snails per plot of either species. No difference in inflicted damage to various treatments was observed between two species, suggesting their equal damage potential on rice. Meanwhile, rice yields in 2 cm water level treatments were compatible with 5 cm control treatment. The least yield was recorded in treatments with three snails per plot of either species at 5 cm water level. Understanding the effect of sowing method and suitable water level is important as it can be further incorporated into rice cultivation practices to reduce damage of apple snails and ensure a high yield during harvest.

The growth parameters of the green lacewing, Chrysoperla nipponensis-B (Okamoto), were studied under laboratory conditions. The highest mortality was recorded in the immature stages (instars 1st, 2nd, 3rd and pupae) of C. nipponensis fed on the eggs of Corcyra cephalonica (37.26%). The sex ratios (proportion of female to male) when reared on the eggs of C. cephalonica and an artificial diet with ginger were 0.93 : 1.00 and 0.87 : 1.00, respectively. The maximum life spans of females reared on the eggs of C. cephalonica and an artificial diet with ginger were 63 and 64 days, respectively. The females reared on the eggs of C. cephalonica produced the highest number of eggs (10.4) on the fifth day of oviposition, whereas on the artificial diet with ginger it was 9.26 on the eighth day of oviposition. The net reproductive rate (Ro) and maximum gross reproductive rate (GRR) of C. nipponensis fed on the eggs of C. cephalonica were 69.50 and 223.10 females per female per generation, respectively, whereas for the artificial diet with ginger they were 117.24 and 236.89 females per female per generation, respectively. Mean generation time (T) was 37.06 and 48.16 for the eggs of C. cephalonica and artificial diet with ginger, respectively. The intrinsic rate of natural increase (r) was 0.11 and 0.09 females per female per day for the eggs of C. cephalonica and artificial diet with ginger, respectively. The finite rate of increase (λ) was 1.12 and 1.11 females per female per day for the eggs of C. cephalonica and artificial diet with ginger, respectively. The population doubling time (DT) was 6.05 days on the diet of eggs of C. cephalonica and 7.00 on the artificial diet with ginger.

This study investigated the effects of the first generation anti-coagulant rodenticide, chlorophacinone and the second generation rodenticide, bromadiolone on nestling growth of barn owl, Tyto alba javanica in oil palm plantations. Forty-two nestlings were selected from rodenticide-free, chlorophacinone and bromadiolone treated plots. Growth rates of nestlings were measured by taking body weight, culmen length, tarsus length and wing length on a periodic basis. Dead nestlings were collected during daily observations. Autopsies were carried out to determine cause of mortality and internal organ (livers) were harvested for analysis of residue by using high performance liquid chromatography (HPLC). Rodenticide-free plot scored the highest fledging success, i.e. 71.4% (n = 14) at 52 days after hatching. In chlorophacinone treated plot, six out of 14 nestlings or 42.85% successfully grew to fledging age of 52 days. In bromadiolone treated plot, only five or 35.75% (n = 14) of nestlings survived to fledging age. Three out of nine nestling carcasses found in bromadiolone treated area were collected for the analysis. Of these, two were detected with bromadiolone residue measuring 0.33 μg g-1 and 0.41 μg g-1 wet weight, respectively. In chlorophacinone treated plot, three out of eight carcasses were collected for analysis. The residue of chlorophacinone detected ranged from 0.18 μg g-1 to 0.21 μg g-1. Nestlings that survived in the rodenticide-free area showed higher measurements for body weight, tarsus, culmen and wing length compared to bromadiolone and chlorophacinone treated plots.

Helopeltis antonii is one of the major pests in most cashew growingareas in Indonesia. The pest attacks cashew plants in the nursery and alsoin the fields which caused significant damage. Apart of cashew the insectshave a lot of alternate host plants such as tea, cocoa, neem, pepper,mangoes, jamboes, etc. This research was aimed at investigating thedispersion pattern of H. antonii in cashew plantation. The research wasconducted in smallholder cashew plantation in Ngadirejo, Wonogiri,Indonesia from March 2004 to May 2006. The observation of H. antoniipopulation was carried out on 60 sample plants which selectedsystematically in about 2 ha cashew plantation area every two weeks.Distribution analysis using various indices of dispersion and regressionmodels was applied to evaluate the dispersion of H. antonii in cashewplantation. The result shows that variance to mean ratio (s 2 / x ), χ2, Lloydmean crowding, Green’s coefficient of dispersion and Taylor’s power lawindicate aggregated distribution when the population is high duringflushing-flowering seasons of cashew plants, and it indicates regular orrandom distribution when the population is low during post-floweringseasons. The aggregated distribution on cashew plants indicated that thereis a preferency to food sources of the plants and an individual behaviour toaggregate. Therefore, sampling and monitoring H. antonii in cashewplantation should be carried out systematically during flushing-floweringseasonsKey words: Cashew, Anacardium occidentale, pest management,Helopeltis antonii, dispersion, Central JavaABSTRAKPola sebaran Helopeltis antonii Signoret (Hemiptera:Miridae) pada pertanaman jambu meteHelopeltis antonii merupakan salah satu hama utama tanamanjambu mete yang ditemukan hampir di setiap area pengembangan mete diIndonesia. Hama ini menyerang tanaman jambu mete sejak pembibitanhingga di lapangan dengan kerusakan yang cukup signifikan. Selain jambumete, serangga ini juga mempunyai banyak inang alternatif antara lain teh,kakao, mimba, lada,mangga, jambu air dll. Penelitian ini bertujuan untukmenentukan sebaran serangga tersebut, khususnya sebaran horizontal padapertanaman jambu mete. Penelitian dilakukan di pertanaman jambu metemilik petani di daerah Ngadirejo, Wonogiri, Indonesia dari Maret 2004sampai Mei 2006. Pengamatan populasi H. antonii dilakukan pada 60tanaman sampel yang dipilih secara sistematik pada area pertanamanjambu mete seluas kurang lebih 2 ha setiap 2 minggu sekali. Hasil analisismenggunakan beberapa indek pengelompokan dan model regresi yaiturasio keragaman terhadap rata-rata (s 2 / x ), χ2,rata-rata pengelompokandari Lloyd, koefisien sebaran dari Green dan hukum kekuatan Taylormenunjukkan penyebaran yang mengelompok jika populasi tinggi selamamusim pembentukan tunas dan pembungaan jambu mete, dan merata atauacak jika populasi rendah sesudah musim pembungaan. Pengelompokan H.antonii pada tanaman jambu mete menunjukkan adanya ketertarikan padaketersediaan makanan pada tanaman jambu mete dan perilaku individuserangga tersebut. Oleh karena itu untuk pengambilan sampel danpengamatan serangga tersebut sebaiknya dilakukan secara sistematis padasaat musim pembentukan tunas dan pembungaan.Kata kunci: Jambu mete, Anacardium occidentale, pengendalian hama,Helopeltis antonii, sebaran, Jawa Tengah

This study aimed to improve the efficacy of azadirachtin (Azadirachta indica. A. Juss) against two serious pest species of stored products, Sitophilus oryzae (L.) and Tribolium castaneum (Herbst), through nano-emulsion formulations. Pseudoternary phase diagrams were constructed consisting of an emulsion system of an active ingredient (neem oil), surfactant (polysorbate or alkylpolyglucoside), and water. Isotropic regions were formed in the pseudoternary phase diagrams, and four formulations were selected from the isotropic regions and characterized according to particle size, particle aging, zeta potential, stability and thermostability, surface tension, viscosity, and pH. The selected formulations showed particle sizes of 208–507 nm in diameter. The result of contact toxicity demonstrated excellent mortality of S. oryzae and T. castaneum adults, with a mortality range of 85–100% and 74–100%, respectively, at a 1% azadirachtin concentration after only 2 days of exposure. Compared to non-formulated neem oil, the nano-emulsion formulations significantly increased the mortality of the tested species.

Achroia grisella, is a noxious pest of honey bee hives. In this study the toxicity of honey bee venom and bacterial Cry1C toxin of Bacillus thuringiensis on A. grisella were assessed. In addition, the synergistic interaction between BT Cry1c and crude honey bee venom was determined. The combination of HBV and Cry1C increased the activity for both toxins. Therefore, further analysis was carried out to assess the synergistic interaction of each compound assay separately and in mixture. The results showed LC50 value of 1.105 μg/μl for HBV and LC50 value of 0.201 μg/μl for Cry1C. The combination bioassay result showed an LC50 value of 0.549 μg/μl. The combined calculated LC50 is containing lower concentration of the component toxins, this result proved that the toxicity was enhanced due to the combination. A chi square value of 39.93and relative synergistic factor ratio of 1.14 confirmed that a synergic interaction was achieved in combination.

Imidacloprid (1-[(6-chloro-3-pyridinyl)methyl]-N-nitro-2-imidazolidinimine), with a novel mode of action is a recent systemic and contact insecticide with high activity against a wide range of pests. Continuous dispersion of this pesticide in the environment and its stability in soil results in environmental pollution which demands remediation. The present research was attempted to isolate and characterize imidacloprid degrading bacteria from vegetable farms of Cameron Highlands in Malaysia. The degradation ability of the isolates was tested in minimal salt medium (MSM) for a duration of 25 days and the selected strains were characterized based on their biochemical and molecular characteristics. Levels of imidacloprid in MSM medium were analyzed by high performance liquid chromatography (HPLC). Among 50 soil bacterial isolates Bacillus sp., Brevibacterium sp., Pseudomonas putida F1, Bacillus subtilis and Rhizobium sp. were able to degrade 25.36–45.48% of the initial amount of imidacloprid at the concentration of 25 mg L−1 in C limited media. Brevibacterium sp. was isolated from organic farms that had never been exposed to imidacloprid while the other farms had previously been exposed to different levels of imidacloprid. All bacteria introduced in this study were among the first reports of imidacloprid degrading isolates in C limited media from tropical soil. Therefore, the results of this study demonstrate the effectiveness of using soil bacteria for microbial degradation of imidacloprid. These findings suggest that these strains may be promising candidates for bioremediation of imidacloprid-contaminated soils.

The aim of the present study was to investigate the virulence of the entomopathogenic fungus Metarhizium anisopliae (isolates PR1 and GT3) under osmotic stress condition. The virulence study of the fungus was conducted by three ways—growth (germination, vegetative growth and sporulation); enzymatic activities (chitinase, protease and lipase) of M. anisopliae and percentage mortality of Bemisia tabaci to M. anisopliae. Conidia of M. anisopliae were produced under different osmotic stress conditions as SDA medium as control, SDA medium with 0.5 M NaCl, SDA medium with 0.5 M KCl, SDA medium with 1 M NaCl and SDA medium with 1 M KCl. The germination percentage, vegetative growth, sporulation, chitinase and protease activities were highest for control of PR1 isolate, reaching up to 97 %, 4.1 cm and 6.6 × 106 conidia/ml, 2.6 mU/ml and 1.7 µg/ml/min, respectively. These values decreased up to 86.7 %, 3.6 cm and 4.1 × 106 conidia/ml, 1.6 mU/ml and 1.0 µg/ml/min, respectively under osmotic stress. The lipase activity was highest for 0.5 M NaCl of PR1 isolate, reaching up to 18.2 µmol/ml/min. The mortality percentage of B. tabaci was highest for control of PR1 and GT3 isolates, reaching up to 83.9 and 83.8 %, respectively. These values decreased up to 77.4 and 77.5 %, respectively under osmotic stress. This paper concludes that both the isolate PR1 and GT3 are virulent to B. tabaci under osmotic stress condition.

A rapid and simple method for the determination of glyphosate in crude palm oil (CPO) was developed and validated using high performance liquid chromatography with fluorescence detector. Glyphosate was derivatized with 9-fluorenylmethylchloroformate (FMOC-Cl) and then separated using a C18 reverse phase column with potassium dihydrogen phosphate and acetonitrile as the mobile phase. A linear correlation was obtained for the concentration of glyphosate from 0.05-1.5 μg mL-1 with a correlation coefficient of 0.9998. The average recovery obtained for glyphosate ranged between 80% and 100% at five fortification levels with the relative standard deviation (RSD) of less than 3% of all cases. The limit of detection and limit of quantification for glyphosate were 0.05 and 0.1 μg/g, respectively. The method will facilitate palm oil trade through quality assurance in terms of glyphosate residues in palm oil products and also to counter any issues related to food safety for palm based products.

This study investigated the effects of the first generation anticoagulant rodenticide chlorophacinone and the second generation rodenticide bromadiolone on the population and breeding performances of barn owls at oil palm plantations. Three treatment plots were established: one baited with chlorophacinone, one with bromadiolone, and the third kept rodenticide-free. Four rat-baiting campaigns which coincided with barn owl breeding season were carried in the rodenticide-treated plots. The occupancy rate of nest boxes, clutch size, brood size and fledging rates of the barn owls in each plot were monitored weekly throughout the study. Freshly regurgitated pellets from barn owls were collected from all occupied nest boxes at weekly intervals during the development of nestlings in each breeding season. The results show that the occupancy rate of the nest boxes was significantly higher in the rodenticide-free area compared with both rodenticide-treated areas. Similarly, the breeding performances, such as clutch size, brood size and fledging success, were higher in the rodenticide-free area compared with the rodenticide-treated areas. Results of high performance liquid chromatography (HPLC) analysis showed that 20.56% (mean residue: 1 .335 ± 0.073 lg/g) and 28.89% (mean residue: 0.777 ± 0.032 lg/g) of the collected regurgitated pellet samples from the rodenticide-treated areas contained bromadiolone and chlorophacinone residue, respectively. The mean brood size and fledging success of the barn owls showed a strong negative correlation with the mean concentration of rodenticide residues present in the regurgitated pellets and with the percentages of pellets detected having such residues (R2 k 0.44, P < 0.05). Similarly, the mean clutch size of barn owls was negatively correlated with the mean concentration of rodenticide residues and with the percentages of pellets detected with these residues. However, the correlation was not significantly different (R2 k 0.34, P > 0.05). In general, the higher the amount of residues detected, the lower the breeding performance parameters measured in this study

Piper sarmentosum is a cultivated plant that also grows wildly in the South East Asian region. It is well known for its medicinal properties and a variety of active chemical constituents. The control potential of the plant extract against bacterial leaf blight caused by Xanthomonas oryzae and sheath brown rot of rice caused by Pseudomonas fuscovaginae was evaluated. The aqueous methanol extracts of fruits and leaves of P. sarmentosum were chemically analyzed using gas-chromatography mass-spectrometry (GC-MS). The results gave the highest best matches of 28 compounds (80.2% of total extracts) in the leaf extract and 24 compounds (89.1% of total extracts) in the fruit extract. The flavonoid fraction 4H-pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- (DDMP) was found to be the major component which constitutes a complex natural product that has been of recent medicinal interest. The extract of the fruit had IC50 of 28.08 and 8.41 mg ml−1 against P. fuscovaginae and X. oryzae, respectively, while the leaf extract had IC50 of 10.42 and 24.69 mg ml−1 against P. fuscovaginae and X. oryzae, respectively.

The aim of this study was to investigate the molecular identification and virulence of the entomopathogenic fungus Metarhizium anisopliae. Initially, the alignments of nucleotide sequences of the internal transcribed spacer (ITS) 1-5.8S-ITS4 regions of six isolates–TFFH1, TFFH3, PR1, MAP, GT3, and GT2–were investigated using GenBank ITS sequences for the same region and species of fungus. Thereafter, the in vitro (germination, vegetative growth, and sporulation) and the in vivo (%mortality of Bemisia tabaci) virulence of the fungus were investigated. A BLAST search of the ITS1 region revealed a match to a sequence registered under the accession number for M. anisopliae. The submitted sequence data were registered and provided with the accession numbers JX041507 to JX0415012, which were released in August 2012 (GenBank). In the virulence experiments, the highest germination percentage, vegetative growth, and sporulation of M. anisopliae were observed in the isolate PR1, with mean values of 97.2%, 4.6 cm, and 7.2 × 106 conidia/ml, respectively; while the highest mortality percentage of B. tabaci was observed in the isolate GT3, with an mean value of 84.3%. This study concludes that all the six tested isolates–TFFH1, TFFH3, PR1, MAP, GT3, and GT2–are M. anisopliae and the isolates PR1 and GT3 are more virulent to B. tabaci than that by the other four isolates.

Bemisia tabaci, a resistance-prone insect pest, is a cryptic species complex with important invasive biotypes such as B and Q. The biotype and resistance statuses of this pest in Malaysia remain unclear. This study assessed the biotype and resistance status of a number of contemporary populations of B. tabaci based on the mtCO1 marker and the dose-response method, respectively. The Pahang (PHG) population was labelled as the Q biotype, while the remainder of the populations belonged to the Asia 1 biotype. A very low level of resistance for profenofos, cypermethrin, and imidacloprid was detected for all populations [resistance factor (RF) 100). All populations showed a very low level of resistance against pymetrozine except Q-type PHG population, which exhibited a very high level of resistance. For most insecticides, the highest level of resistance was detected in the PHG population. The implications of these findings for better management of this noxious pest are discussed.

Herbicidal potential of aerial parts of Tinospora tuberculata on germination and seedling growth of seven test plant species, namely rice (Oryza sativa L.); two rice weeds, barnyardgrass (Echinochloa crus-galli L.) and weedy rice (O. sativa f. spontanea); and four vegetable crops, lettuce (Lactuca sativa L.), tomato (Solanum lycopersicum L.), carrot (Daucus carota L.), and cucumber (Cucumis sativus L.) were evaluated. Six concentrations of methanol extract (3.12, 6.25, 12.5, 25, 50, and 100 g L−1) were compared with the control (distilled water). The rate of seed germination and the radicle and hypocotyl length of 7-day-old test plant seedlings were reduced as the concentration of extracts increased compared to the control. Generally, the degree of toxicity of extracts derived from the leaves was more than the extracts derived from the stem. Cluster analysis and the concentrations required for 50% inhibition (defined as EC50) of all parameters showed that radicle growth was more suppressed than germinatio...

Aim: This study aims to understand the distribution of Pomacea spp. in various rice fields of Peninsular Malaysia. Place and Duration of study: The study was conducted at twenty five rice fields from five states in Peninsular Malaysia (Selangor, Perak, Penang, Kedah and Kelantan) during the sowing and harvesting of off rice season-2013. Methodology: This study was carried out on rice fields of one acre in size which were further divided into four blocks. A 0.5m quadrate was used for sampling and snails within the quadrate were collected, counted and identified based on their shell morphology by the experts. Results: Based on this study, Pomacea maculata was more abundant and widely distributed as compared to Pomacea canaliculata. Rice fields in Perak showed the highest population distribution of Pomacea spp. during both sowing and harvesting of rice. Original Research Article Annual Research & Review in Biology, 4(24): 4123-4136, 2014 4124 Meanwhile, the lowest population was observed in rice fields during the sowing of rice in Kelantan and during harvesting of rice in Kedah. Comparatively higher population of Pomacea spp. was recorded during sowing of rice than harvesting. No population of P. canaliculata was observed from the rice fields of Penang state. Water temperature, pH and the number of rice seedlings does not have a significant effect on the distribution of Pomacea spp. Conclusion: The results from this study could help in an area wide management program against Pomacea spp., not only to minimize their damage to rice but also to control their further spread.

Children are the vulnerable group in the agricultural community due to their early exposure to pesticides through the dynamic interplay between genetic predisposition, environment, and host-related factors. This study aims to identify the possible association between the depression in blood cholinesterase level and genotoxic effect among farm children. The results of micronuclei assay and comet assay showed that the reduced blood cholinesterase level from organophosphate pesticide exposure is significantly associated with an increase in chromosome breakage and DNA strand breaks. These genotoxicity end points suggest that farm children's cells experience early DNA damage that may lead to uncontrolled cell proliferation during their adulthood. Thus, farm children who grow up near pesticide-treated farmland have a higher probability of developing cancer than children with minimal or zero exposure to pesticides.

Metisa plana (Walker) is leaves defoliating insect that is able to cause a staggering loss of USD 2.32 billion within two years to Malaysian oil palm industry. Therefore, an early warning system to predict the outbreak of Metisa plana that is sustainable in terms of cost, time, and energy is crucial. Nonetheless, the current approaches of conventional practices are highly dependent on ineffective and time-consuming in-situ data collection. Geospatial technologies can be used to obtain data in rapid, harmless, and cost-effective manners. Hence, this study utilized the technologies such as land surface temperature (LST), rainfall (RF), relative humidity (RH), and Normalized Difference Vegetation Index (NDVI) to i) examine climatic stresses that cause the outbreak of Metisa plana, ii) to construct the relationship between geospatial data and Metisa plana outbreak, and iii) to predict the outbreak of Metisa plana in oil palm plantation. LST between 24°C and 28°C showed a strong relationship with the presence of Metisa plana. Consistent day pattern was absent in the correlation between LST, RF, RH with Metisa plana. Presence of Metisa plana was not correlated with NDVI. ANN prediction models with the highest accuracy of 95.42% was achieved by using RH data. Model generated by combined variables were Able to predict the presence of Metisa plana with the accuracy of 72.64%. In summary, the elucidation of Metisa plana's landscape ecology is possible with the utilization of geospatial technology, and temperature has been found to be the most important factor that influence the presence of Metisa plana.

The excessive application of pesticides for agricultural production in Malaysia has raised quite some concern about environmental safety and sustainability. To reduce environmental impact of pesticide overuse, there is an increasing interest in using different elicitors to induce disease resistance in crop. Chilli (Capsicum annuum L.), which is an important vegetable cum spice crop in Malaysia, was used to compare the effectiveness of two natural elicitors (jasmonic acid and salicylic acid) with conventional pesticide application as control. The experimental results indicated that pesticide-treated plants showed rapid reduction in disease severity after application while elicitors perform slowly and its effectiveness increase gradually over time. Among the tested elicitors, jasmonic acid was found most effective regarding disease severity and yield of chilli compared with salicylic acid. Although used elicitors was not best performing treatment compared with conventional pesticide, some physiological parameters (relative chlorophyll content, chlorophyll fluorescence and photosynthesis rate) and disease severity in chilli plants treated with jasmonic acid was very close to conventional pesticide. Therefore, jasmonic acid could be a potential elicitor for inducing disease resistance in chilli and salicylic acid may not an appropriate elicitor for chilli.

Anthracnose is an economically important disease that can cause 10 to 80% yield reduction in the market with its typical symptoms of dark spots, sunken necrotic tissues and concentric rings of acervuli on fruits. One of the important methods to control the disease is by using antifungal agents derived from plant. Preliminary study indicated that the extracts of Allamanda species have the potential to be developed as the target agent. Five Allamanda species were extracted and screened for antifungal activity against plant pathogenic fungus Colletotrichum gloeosporioides by using poison agar technique. The three chloroform extracts of A. blanchetti, A. cathartica ‘Alba’ and A. cathartica ‘Jamaican Sunset’ exhibited potent inhibitory effects and suppressed the mycelial growth of C. gloeosporioides by up to about 70%. Further study by bioautography-guided fractionation of the extracts led to the isolation and identification of tetracyclic sesquiterpene plumericin as the antifungal agent. The structural determination of the compound was carried out by interpreting the IR, MS, 1D-NMR and 2D-NMR spectral data.

Carica papaya is a member of the Caricaceae. Its leaves have been used in folk medicine for centuries. Recent studies have shown its beneficial effects as an anti-inflammatory agent (Owoyele et al 2008) and anti-tumour15 as well as antioxidant and wound healing properties7. The study has shown that the effect of carica papaya leaves juice intake also can accelerate the rate of increase in platelet count among the patients infected with dengue fever and dengue haemorrhagic fever18. With all the goodness of carica papaya leaves, a formulation with addition of virgin coconut oil (VCO) is produced to give an enhanced supplement beverage to market nowadays. Virgin coconut oil is well known as anti-oxidant4. The combination of these two substances gives a balance combination in healthy supplement. In recent years the application of emulsion is rapidly increasing in many fields such as cosmetics and paints. Emulsions are dispersions of droplets of one liquid in another, immiscible, liquid in which the droplets are of colloidal or near-colloidal sizes. The combination of water and oil (VCO) with addition of non-ionic surfactant Tween80 was constructed using ternary phase diagram. By considering the Hydrophilic-Lipophilic Balance (HLB) value of each substance will help in producing a stable emulsion.

BACKGROUND: Pesticides are developed with carriers to improve their physicochemical properties and, accordingly, the bioefficacy of the applied formulation. For foliar-applied herbicide, generally less than 0.1% of the active ingredient reaching the target site could reduce pesticide performance. Recently, a carrier of nanoemulsion consisting of oil, surfactant and water, with a particle size of less than 200 nm, has been shown to enhance drug permeability for skin penetration in pharmaceutical delivery systems. In the present work, the aim was to formulate a water-soluble herbicide, glyphosate isopropylamine (IPA), using a green nanoemulsion system for a biological activity study against the weeds creeping foxglove, slender button weed and buffalo grass. RESULTS: The nanoemulsion formulations displayed a significantly lower spray deposition on creeping foxglove (2.9–3.5 ng cm−2), slender button weed (2.6–2.9 ng cm−2) and buffalo grass (1.8–2.4 ng cm−2) than Roundup® (3.7–5.1 ng cm−2). The visible injury rates of weeds treated with the nanoemulsion formulations were statistically equivalent to those relating to Roundup® at 14 days after treatment, with a control range of 86.67–96.67%. CONCLUSION: It was hypothesised that the significant difference in spray deposition with equal injury rates can be attributed to enhanced bioactivity of the nanoemulsion formulations. This initial discovery could be the platform for developing better penetration of agrochemical formulations in the future. Copyright © 2012 Society of Chemical Industry

Two extraction methods were successfully applied to study the extraction of imidacloprid from soil. The first method, using a mix of acetone and hexane, was based on Soxhlet extraction, and the second method, using acetonitrile, methanol, and water, was a modified version of a liquid extraction method. Quantification was performed by reversed-phase High Performance Liquid Chromatography (HPLC) with Diode Array Detection (DAD) at 270 nm using 40:60 (v/v) acetonitrile/water as a mobile phase. The mean recoveries for imidacloprid from soil ranged from 82.6 to 109%, with a relative standard deviation between 1.9 and 5.6% for both extraction methods. The detector linearity and the reproducibility of the method proved to be very precise. The limits of detection were 0.08 and 0.06 mg kg−1 for liquid extraction and Soxhlet extraction, respectively. Overall, the efficiency of the Soxhlet extraction at lower concentrations was better than at higher concentrations, while liquid extraction proved efficient for all sp...