Visual detection of <Emphasis Type="Italic">Didymella bryoniae</Emphasis> in cucurbit seeds using a loop-mediated isothermal amplification assay

A method was developed using a Loop-mediated isothermal amplification assay (LAMP) for detecting Didymella bryoniae in cucurbit seeds. The LAMP primers were designed based on the DNA-dependent RNA polymerase II RPB140 gene (RPB2) from D. bryoniae. Calcein was used as an indicator for the endpoint visual detection of DNA amplification. The LAMP assay was conducted in isothermal (65 °C) conditions within 1 h. The detection threshold of the LAMP assay was 10 pg of genomic DNA and D. bryoniae was detected in 100 % of artificially infested seedlots with 0.05 % infestation or greater. With the LAMP assay, 16 of 60 watermelon and muskmelon seedlots collected from Xinjang province were determined to be positive for D. bryoniae. In contrast, a real-time PCR assay determined that 11 of the 60 seedlots from Xinjiang province were positive for the pathogen. These results showed that the LAMP technique was simple, rapid and well suited for detecting D. bryoniae DNA, especially in seed health testing.     

Vertical distribution of resting spores of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis> in soil

Pratylenchus zeae parasitizes various crops and damages the host roots, resulting in decreased yield and quality of the host plants. Alignments of mitochondrial DNA (mtDNA) Cytochrome Oxidase I (COΙ) sequences revealed the genetic variation among Pratylenchus species. The results indicated 0.2–2.4% intraspecific variations for mtDNA COI sequences among eight P. zeae populations, and 25.4–35.1% interspecific variations between P. zeae and other Pratylenchus species. Based on the mtDNA COΙ region, a loop-mediated isothermal amplification (LAMP) assay was developed for the rapid and specific detection of P. zeae. The optimal conditions for the LAMP assay were 64 °C for 40 min. The LAMP products were confirmed using conventional polymerase chain reaction (PCR), analysis with the restriction enzyme Bam HI and visual inspection by adding SYBR Green I to the products. The LAMP assay could detect P. zeae populations from different hosts and different geographical origins specifically. The LAMP assay was also sensitive, detecting 0.1 individual P. zeae, which was 10 times more sensitive than conventional PCR. This is the first report of the detection of Pratylenchus spp. using LAMP. In addition, the results also suggested that use of the COI gene might allow for good resolution at the Pratylenchus species level.     

Rapid diagnosis of soybean anthracnose caused by <Emphasis Type="Italic">Colletotrichum truncatum</Emphasis> using a loop-mediated isothermal amplification (LAMP) assay

A loop-mediated isothermal amplification (LAMP) assay that directly detects Colletotrichum truncatum in diseased soybean tissues is described, thus allowing rapid diagnosis of soybean anthracnose. Using the target gene Rpb1 (that codes for the large subunit of RNA polymerase II), we designed and screened a set of species-specific primers allowing amplification at 62 °C over 70 min. After addition of SYBR Green I to the LAMP reaction products, a yellow-green color (visible to the unaided eye) developed only in the presence of C. truncatum. The detection limit of the LAMP assay was 100 pg (per μL genomic DNA). The Rpb1-Ct-LAMP assay has been successfully used to diagnose soybean anthracnose in field samples collected from Jiangsu, Anhui and Hubei provinces of China, and to detect C. truncatum in soybean seeds from farmers’ markets. Our results show that the Rpb1-Ct-LAMP assay is a useful and convenient method for detecting C. truncatum, and thus for diagnosis of soybean anthracnose.     

Practical method combining loop-mediated isothermal amplification and bait trap to detect <Emphasis Type="Italic">Pythium helicoides</Emphasis> from hydroponic culture solutions

Two detection methods combining loop-mediated isothermal amplification (LAMP) and a bait trap were developed to detect Pythium helicoides in greenhouses containing roses, miniature roses, and poinsettias in hydroponic culture systems. In “Bait-LAMP”, a crude extract derived from perilla seeds as the bait was used in the LAMP reaction, whereas in the “Bait culture-LAMP”, a crude extract of mycelia grown out from perilla seeds onto Pythium-selective medium served as the bait. The two methods are simple and rapid for practical monitoring of P. helicoides in hydroponic culture systems.     

Rapid and sensitive detection of <Emphasis Type="Italic">Meloidogyne mali</Emphasis> by loop-mediated isothermal amplification combined with a lateral flow dipstick

Meloidogyne mali Itoh, Ohshima & Ichinohe, 1969 is a polyphagous root-knot nematode that infects a wide range of host plants and is subject to phytosanitary restrictions in many countries, including China. In this study, we integrated the loop-mediated isothermal amplification (LAMP) technique with visual detection by a lateral flow dipstick (LFD) and developed an accelerated LAMP-LFD method for the rapid identification of M. mali, targeting the internal transcribed spacer regions and 5.8S ribosomal DNA sequence (ITS-5.8S). The entire test could be performed within 45 min, including 35-min LAMP amplification, 5-min hybridization, and 3–5-min visualization on LFD. The detection limit of this method was 2-pg bulked M. mali gDNA per reaction, and the equivalent of 1‰ single female adult or 1% single second-stage juvenile (J2) could be detected. It is recommended that DNA is extracted from single J2 isolated from soil samples by the modified Baermann funnel method or from an adult female from galls. Based on this, the field sensitivity and specificity of this LAMP-LFD method for the detection of M. mali in field soil samples were both 100% compared to traditional microscopic observation. These results indicate that the LAMP-LFD method is rapid, sensitive, accurate, and simple, and would be applicable for the routine monitoring of phytosanitary M. mali.     

A loop mediated isothermal amplification (LAMP) assay for rapid and reliable detection of <Emphasis Type="Italic">Anguina wevelli</Emphasis>, a grass parasitic nematode

Anguina wevelli is a pathogenic grass parasitic nematode, however it is difficult to identify based simply on morphology. This study developed a loop-mediated isothermal amplification (LAMP) assay to detect A. wevelli. The LAMP method developed could specifically detect A. wevelli in 45 min, and the detection limit was 1/80000 of the total DNA extracted from a single juvenile (J2), an equivalent of 2.5 pg of DNA. This is the first report of the detection of Anguina spp. by using a LAMP-based method.     

Fast detection by loop-mediated isothermal amplification (LAMP) of the three begomovirus species infecting tomato in Panama

Potato yellow mosaic Panama virus (PYMPV), Tomato leaf curl Sinaloa virus (ToLCSiV) and Tomato yellow mottle virus (TYMoV) of genus Begomovirus (family Geminiviridae) are the only three begomovirus species detected infecting tomato (Solanum lycopersicum L.) in Panama. PYMPV, ToLCSiV and TYMoV induce symptoms of stunting, yellowing, curling, distortion of leaves and reduction of fruit size and cause important economic loses. A loop-mediated amplification under isothermal conditions (LAMP) assay was developed for the individual detection of these three begomovirus species by using a set of three primer pairs specific per each one of them. Amplification products were visualized by gel electrophoresis or direct Gel-Red staining of DNA into the reaction tube. PYMPV, ToLCSiV and TYMoV were detected in total DNA extracts obtained from different plant tissues such as leaves, stems, flowers, fruits and roots of infected tomato plants collected in different production regions of Panama. LAMP sensitivity was similar to that of conventional PCR but, the first procedure was faster and cheaper than the last one. Moreover, all three viruses were successfully detected by LAMP and not by conventional PCR from sap extracts obtained from leaf tissues of infected tomato plants which were embedded into 3MM Whatman paper and stored several days, facilitating the samples processing as well as the material movement among different laboratories. Therefore, LAMP is a specific, rapid and cheap procedure to detect all three begomoviruses infecting tomato in Panama and it is suitable for field surveys and sanitation programs.     

Fast and sensitive on-site isothermal assay (LAMP) for diagnosis and detection of three fruit tree phytoplasmas

Over the years, real-time PCR outflanked endpoint PCR in phytopathogen diagnostics, mainly because of the increase in sensitivity and timesaving aspects of the technique. However, a time consuming 16S rRNA-based nested PCR method is still the gold standard for phytoplasma diagnosis. This is also the case for phytoplasma detection in Malus, Pyrus and Prunus, the three main host plants of apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) phytoplasma, respectively. The last decade, loop-mediated isothermal amplification (LAMP) (Notomi et al. 2000) is gaining a lot in significance and is also for phytoplasmas expected to become a widely used reliable diagnostic tool. High specificity and sensitivity which also requires a less stringent need for DNA purification, and the short analysis time and the limited equipment requirements makes the LAMP method a fast and affordable alternative with great point-of-care diagnostic potential. In this paper, we present a LAMP primer set for the ribosomal group 16SrX, containing the important fruit tree phytoplasmas AP, PD and ESFY. The primers were developed and validated for fast and sensitive detection and general use for diagnosis. We foresee that the LAMP technique will also have its application in on-site diagnosis of the fruit tree phytoplasmas during inspections and surveys.     

Development of a loop-mediated isothermal amplification assay for sensitive and specific detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">cucumerinum</Emphasis> Owen

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum Owen (FOC), is a destructive disease affecting cucumber production worldwide. Developing an accurate and reliable method for detection of FOC is important for disease prediction and control. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for specific and sensitive detection of FOC. Four LAMP primers were designed based on the sequence of the FOC-specific random amplified polymorphic DNA (RAPD) marker OPZ-12₈₆₅. LAMP reactions were performed at different temperatures and for different durations, and the optimal temperature and duration were 63 °C for 60 min, respectively. Hence, a LAMP assay for detection of FOC was established. The specificity of the LAMP method was evaluated against 119 isolates of FOC and other pathogens, and only FOC isolates yielded positive results. In sensitivity tests, the lowest concentration of genomic DNA required for the LAMP assay was 10 fg in a 25 μL reaction. The LAMP assay was successfully applied to detect FOC in cucumber tissues and soil from infested fields, and the positive ratios of LAMP, PCR, and traditional tissue isolation for detecting FOC from diseased cucumber root samples were100%, 86.6 and 83.3%, respectively. Therefore, the LAMP assay developed herein should serve as a simple, cost-effective, rapid, highly specific, and sensitive tool for the visual detection of FOC and contribute to improved disease management.     

Lethal and sublethal effects of three insecticides on the aphid parasitoid, <Emphasis Type="Italic">Lysiphlebus fabarum</Emphasis> Marshall (Hymenoptera: Aphidiidae)

Lysiphlebus fabarum Marshall is the main parasitoid of the black bean aphid, Aphis fabae Scopoli. Lethal and sublethal effects of insecticides, thiacloprid+deltamethrin, pirimicarb and pymetrozine were evaluated on the parasitoid under laboratory conditions. One-day-old mummies were exposed to the recommended field concentration of either insecticides via dipping method. Adult emergences were reduced by 82.67, 19.98 and 10.67 % for thiacloprid+deltamethrin, pirimicarb and pymetrozine treatments, respectively. Thiacloprid+deltamethrin had the most adverse effect on the fecundity of the emerged females, while pirimicarb and pymetrozine did not have such effects. According to International organization for biological control (IOBC) insecticide toxicity classification, thiacloprid+deltamethrin resulted to be moderately harmful (E = 97.39%), whereas pirimicarb (E = 15.78%) and pymetrozine (E = 5.15%) were harmless. Thiacloprid+deltamethrin negatively affected five of the estimated demographic parameters (GRR, R ₀ , r _m , λ and T ). Pirimicarb negatively affected GRR, R ₀ and T, while it had no adverse effects on r _m and λ. None of the studied demographic parameters were affected by pymetrozine. Our results suggest that pirimicarb and pymetrozine can be considered as safe for L. fabarum, but that thiacloprid+deltamethrin can have serious detrimental of this parasitoid in the field.     

Pathogenicity and characterization of <Emphasis Type="Italic">Colletotrichum lentis</Emphasis>: A causal agent of anthracnose in common vetch (<Emphasis Type="Italic">Vicia sativa</Emphasis>)

A series of studies were carried out on Colletotrichum lentis which had been been identified in 2015 based largely on the distinctive shape of conidia and ITS sequences, and which has been causing severe anthracnose disease symptoms on common vetch plants (Vicia sativa) in Gansu Province in the northwest region of China. A key focus of the present studies was to determine how vetch crops become infected. The addition of residues from harvested common vetch crops to land being prepared as a seedbed was shown to result in the highest levels of disease severity indicating that this management practice was the most likely way for crops to become severely infected. Seed transmission was unlikely to be the cause of severe outbreaks as less than 5% of seeds harvested from severely infected plants carried C. lentis. To verify that the species causing the severe outbreaks of anthracnose disease of vetch crops was C. lentis, sequence analysis of the ITS, TUB2, ACT, HIS3 and GAPDH genes was conducted. C. lentis isolates from common vetch and lentil (Lens culinaris) formed a distinctive group among Colletotrichum species, including those species that infect other forage and field crops. The unique shape of conidia of C. lentis, straight with only one end curved, was confirmed as being reliable for rapid identification of disease outbreaks caused by this damaging fungal pathogen.     

Molecular analysis of <Emphasis Type="Italic">Cercospora beticola</Emphasis> isolates for strobilurin resistance from the Central High Plains,USA

Cercospora leaf spot (CLS), caused by Cercospora beticola, is the most destructive foliar disease and is a problem in sugar beet production areas, such as Central High Plains (states of Colorado, Montana, Nebraska and Wyoming) in the United States. The disease can be controlled by strobilurin fungicides, referred to as quinone outside inhibitors (QoIs), with a single target site on C. beticola. Strobilurin resistance has been reported in beet production areas from the United States, including the Central High Plains. Although strobilurin resistance is quantitatively inherited, it is considered that it has low to medium heritability in the population. Effective diagnostic tools are required for the rapid detection of C. beticola strobilurin resistance. The study obtained a partial nucleotide sequence of the C. beticola cytochrome b gene and determined to a putative protein with ~386 amino acid residues. Eighty C. beticola isolates (2004–2011) from the Central High Plains were analyzed for mutations. We found a single nucleotide polymorphic (SNP) site which led to G143A mutation and was present in 2 C. beticola QoI-resistant isolates. Partial sequences obtained from 82 C. beticola QoI-sensitive isolates showed identical cytochrome b gene. We developed a PCR-RFLP assay that involved an in vitro digestion using Fnu4HI restriction enzyme for the rapid molecular detection of G143A mutation in the C. beticola population. Results indicated the PCR-RFLP assay was reliable, sensitive, and can be used for the rapid detection of C. beticola strobilurin resistance.     

Fungicidal activity of Thai medicinal plant extracts against <Emphasis Type="Italic">Alternaria brassicicola</Emphasis> causing black spot of Chinese kale

Crude ethanol extracts and six organic solvent fractions of 10 Thai medicinal plants were evaluated for their antifungal activity against Alternaria brassicicola in laboratory and under greenhouse conditions. The results showed that the ethanol extracts of Coscinium fenestratum, Piper betle, Syzygium aromaticus and Zingiber cassumunar displayed complete mycelial growth inhibition of A. brassicicola at a concentration of 0.1%. Meanwhile, the crude ethanol extract and methanol fraction obtained from the stems of C. fenestratum revealed the greatest inhibition against A. brassicicola at 10%, forming inhibition zones 2.55–2.58 cm in diameter. In the greenhouse experiments, crude ethanol extracts of C. fenestratum and P. betle at 1% significantly (P?<?0.05) reduced the disease incidence at up to 67%, indicating promising preventive and curative activities against A. brassicicola. This activity is similar to that of iprodione, a widely used commercial fungicide. Interestingly, Illicium verum extract showed a greater curative effect (58% disease reduction) than protective effect (47% disease reduction). Because the C. fenestratum extract showed the highest activity against the black spot pathogen both in vitro and under greenhouse conditions, its methanol fraction was further analyzed by spectroscopic techniques. We found that berberine is a key active substance inhibiting mycelial growth of A. brassicicola. The results of this study showed the potential of Thai medicinal plants as alternatives to the use of synthetic fungicides for controlling black spot in Chinese kale caused by A. brassicicola.     

Development of a TaqMan probe-based insulated isothermal PCR (TiiPCR) for the detection of <Emphasis Type="Italic">Acidovorax citrulli</Emphasis>, the bacterial pathogen of watermelon fruit blotch

Watermelon (Citrullus lanatus) is an important crop of the Cucurbitaceae family in fruit production worldwide. During its production, bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Acidovorax avenae subsp. citrulli) is an important limiting factor on the volume and value of crops. This pathogen is known as a seed-borne pathogen, and the infested seeds can be a primary source of inoculum. Hence, a rapid and sensitive method for detecting A. citrulli on seeds would be an important tool in the management of BFB. In this study, we sought to develop a method to detect A. citrulli bacterial cells based on a TaqMan probe-based insulated isothermal PCR (TiiPCR) assay. Firstly, the specific primers and probe were designed based on a specific DNA fragment from the genome of A. citrulli. Then, PCR amplification was performed with the plasmid DNA to adjust the components of the PCR reagents, such as the concentrations of primers, magnesium chloride, and Taq DNA polymerase. Results revealed that 10 copies of plasmid DNA were detectable within the modified reagents by TiiPCR. Moreover, 10 bacterial cells in each reaction tube were detectable at a 100 % detection rate in this condition with a fluorescent signal intensification over 1.8. Based on these results, we concluded that a specific, rapid, and sensitive method based on TiiPCR had been successfully developed to detect bacterial cells of A. citrulli.     

Parasitism of <Emphasis Type="Italic">Psytallia concolor</Emphasis> (Hymenoptera: Braconidae) on <Emphasis Type="Italic">Bactrocera oleae</Emphasis> (Diptera: Tephritidae) infesting different olive varieties

Psytallia concolor (Szépligeti) is a koinobiont endoparasitoid of many Tephritidae larvae, including Bactrocera oleae (Rossi), and has been used in Mediterranean areas for biological control of olive fruit fly by inundative release. The present study evaluates the influence of olive fruit variety (Amfissis, Arbequina, Branquita de Elvas, Carolea, Kalamon, Koroneiki, Leccino, Manzanilla, Mastoidis, Moroccan Picholine and Picholine) on P. concolor parasitism efficiency and performance in the field during two successive years. The results showed that the percentage of parasitism was significantly higher (>30%) in Mastoidis and Koroneiki (light-weight varieties <1.5 g) than Leccino which has a medium fruit weight, followed by Amfissis, Moroccan Picholine, Picholine and Branquita de Elvas. Only Manzanilla among large weight varieties, exhibited high percentage of parasitization (42.72%) during 2013. The mean weight of the pupae (>4.21 mg) as well as the length of the developed adult parasitoids (>3.5 cm) in Mastoidis and Manzanilla were significantly higher than these individuals developed from other varieties such as Koroneiki and Kalamon. Finally, the optimal host fruit for P. concolor development seems to be Mastoidis variety with great biological parameters and percentage of parasitism.     

Genetic structure of <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> and <Emphasis Type="Italic">P. teres</Emphasis> f. <Emphasis Type="Italic">maculata</Emphasis> populations from western Canada

Infection by Pyrenophora teres f. teres (Ptt) or P. teres f. maculata (Ptm), the causal agents of the net and spot forms of net blotch of barley, respectively, can result in significant yield losses. The genetic structure of a collection of 128 Ptt and 92 Ptm isolates from the western Canadian provinces of Alberta (55 Ptt, 27 Ptm), Saskatchewan (58 Ptt, 46 Ptm) and Manitoba (15 Ptt, 19 Ptm) were analyzed by simple sequence repeat (SSR) marker analysis. Thirteen SSR loci were examined and found to be polymorphic within both Ptt and Ptm populations. In total, 110 distinct alleles were identified, with 19 of these shared between Ptt and Ptm, 75 specific to Ptt, and 16 specific to Ptm. Genotypic diversity was relatively high, with a clonal fraction of approximately 10 % within Ptt and Ptm populations. Significant genetic differentiation (PhiPT = 0.230, P = 0.001) was found among all populations; 77 % of genetic variation occurred within populations and 23 % between populations. Lower, but still significant genetic differentiation (PhiPT = 0.038, P = 0.001) was detected in Ptt, with 96 % of genetic variation occurring within populations. No significant genetic differentiation (PhiPT = 0.010, P = 0.177) was observed among Ptm populations. Isolates clustered in two distinct groups conforming to Ptt or Ptm, with no intermediate cluster. The high number of haplotypes observed, combined with an equal mating type ratio for both forms of the fungus, suggests that P. teres goes through regular cycles of sexual recombination in western Canada.     

Characterization of culturable gut bacterial isolates from wild population of melon fruit fly (<Emphasis Type="Italic">Bactrocera cucurbitae</Emphasis>) and assessing their attractancy potential for sustainable pest management

The melon fruit fly, Bactrocera cucurbitae (Coquillett) poses a serious threat to cucurbit production worldwide. Its management by conventional means remains difficult due to their proclivity to oviposite in fruit. In view of the increasing environmental safety concerns, use of biocontrol agents for sustainable pest control holds immense potential. Given this, the study intended to identify the cultivable bacteria inhabiting the intestinal tract of adult male and female melon fruit flies (Bactrocera cucurbitae) separately from field-collected population, and to assess the attractiveness of these bacteria to the fly. All selected bacterial isolates were identified and characterized based on morphological, biochemical and 16S rRNA gene sequence. Bacterial community identified in the gut of B. cucurbitae predominantly composed of Enterobacteriaceae followed by Staphylococcaceae, Enterococcaceae, Bacillaceae and Brucellaceae. Further, the laboratory bioassay was employed to examine the attractiveness of the supernatant as well as whole culture broth of 10 different species of bacteria to B. cucurbitae adults. Among these, Klebsiella oxytoca and Citrobacter freundii followed by Bacillus cereus were found highly attractive to fruit flies. The field experiment using supernatant of two bacteria demonstrated that the K. oxytoca was significantly more attractive to female flies followed by C. freundii.     

<Emphasis Type="Italic">RB</Emphasis> and <Emphasis Type="Italic">Ph</Emphasis> resistance genes in potato and tomato minimize risk for oospore production in the presence of mating pairs of <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

Composed mostly of fungivorous species, the genus Aphelenchoides also comprises 14 plant-parasitic species. The most common and devastating, A. besseyi, A. fragariae, A. ritzemabosi and A. subtenuis have been reported on more than 900 plant species. The combination of low inter-specific and high intra-specific morphological variability makes morphology-based identification extremely difficult within this genus, and has led to molecular tools being employed to ensure accurate diagnoses. rDNA markers are widely used for the identification of nematodes while the Cytochrome Oxidase I gene (COI) remains relatively unexplored despite its role as the standard barcode for almost all animal groups. To explore its suitability as a diagnostic tool, we studied a fragment of the mtCOI region of the four main plant-parasitic Aphelenchoides within a phylogenetic framework. We generated 69 mtCOI and 123 rDNA sequences of diverse Aphelenchoides taxa; 67 belong to the main plant-parasitic species including the first mtCOI sequence of A. fragariae and the first mtCOI and 28S sequences of A. subtenuis. mtCOI had a similar success rate for PCR amplification. Phylogenetic trees based on the three studied markers are largely in agreement with one another, validating their use for Aphelenchoides diagnosis; additionally, we were able to locate several misidentified sequences of plant-parasitic Aphelenchoides in existing databases. The concatenated analysis from the three markers resulted in a more robust insight into the phylogeny and evolution of Aphelenchoides, revealing that plant-parasitism has evolved independently at least three times within this genus, presumably from fungal-feeding ancestors.     

Efficacy of <Emphasis Type="Italic">Beauveria Bassiana</Emphasis> for the Management of Economically Important Wireworm Species (Coleoptera: Elateridae) in Organic Farming

Wireworms, the larvae of click beetle species Agriotes lineatus, A. obscurus and A. sputator are serious pests for several field crops. They are considered severe pests of potato tubers and the damages caused by them can be resulted a significant loss especially in organic crop production. Since synthetic insecticides are prohibited in Organic Farming; biological control methods have to be used in organic crop production. In the current study naturalis Beauveria bassianaa strain ATCC 74040 was used both under laboratory and field conditions using wheat and potato crops as a food source respectively. Fungus showed a significant mortality in high number of wireworms boxes (50%) compared to low number of wireworms (17%) and untreated boxes (13%) respectively. However, seed germination showed no effect in all three categories. Field data have shown mixed results when B. bassiana has been applied in a variety of application methods. Maximum infestation (3.99%) was recorded in untreated plots; while significantly lower damage (1.11%) was recorded in whole surface treated plots. During first year all the treatments were significantly different from each other; however, no significant differences were noted between furrows and whole surface applications but were different from control treatment during second year of experimentation. The results showed that the biological control of wireworms by using entomopathogenic fungi, such as B. bassiana is a promising target specific option without disturbing the other communities in the soil.     

Effect of provision of apiaceous flowers associated to foods on the biology of <Emphasis Type="Italic">Coleomegilla maculata</Emphasis>

Coleomegilla maculata DeGeer is an omnivorous lady beetle that feeds on natural or alternative prey and artificial foods, which allows its laboratory rearing for use on augmentative biocontrol. In addition, C. maculata may supplement its diet with pollen and nectar, which helps in its conservation in agroecosystems. This study aimed to evaluate if Apiaceous flowers (Anethum graveolens L. and Coriadrum sativum L.), with and without alternative prey [eggs of Ephestia kuehniella (Zeller) or larvae of Drosophila melanogaster Meigen] or an artificial food (aqueous solution of honey), may guarantee the survival and complete development of the immature and adult stages of C. maculata in the laboratory. The immature stages developed only when Apiaceous flowers were offered with E. kuehniella eggs. The food with only one of the alternative prey (moth eggs or fly larvae) or moth eggs + honey solution resulted in fertile adults; however, the number of eggs/cluster was greater for the foods with E. kuehniella eggs + honey solution, A. graveolens flowers, or only D. melanogaster. Foods comprising only the two Apiaceous species, only the honey solution, or only water resulted in larval development up to a specific instar. Adults of C. maculata also survived on these foods, but there was no oviposition. The foods of the two Apiaceous species produced heavier adults only when associated with E. kuehniella eggs. The results indicate that the zoophytophagous habit of C. maculata should be considered in conservation biocontrol programs aimed at using this lady beetle to control crop pests.