Development of an artificial diet for mass rearing of the spiny bollworm,Earias insulana

An artificial diet for rearing the spiny bollworm,Earias insulana (Boisd.), was developed in experiments with three successive generations. The present recommended diet is based on kidney beans, alfalfa meal, whole powdered milk and yeast; methyl-P-hydroxy benzoate, chloramphenicol and formaldehyde were included as preservatives. The effect of the diets on the insect quality is discussed.     

Expression of esterase gene in yeast for organophosphates biodegradation

Organophosphates are esters of phosphoric acid and can be hydrolyzed and detoxified by carboxylesterase and phosphotriesterase. In this work esterase enzyme (Est5S) was expressed in yeast to demonstrate the organophosphorus hydrolytic activity from a metagenomic library of cow rumen bacteria. The esterase gene (est5S) is 1098 bp in length, encoding a protein of 366 amino acid residues with a molecular weight of 40 kDa. Est5S enzyme was successfully produced by Pichia pastoris at a high expression level of approximately 4.0 g L⁻¹. With p-nitrophenol butyrate as the substrate, the optimal temperature and pH for enzyme activity were determined to be 40 °C and pH 7.0, respectively. The esterase enzyme was tested for degradation of chlorpyrifos (CP). TLC results obtained inferred that CP could be degraded by esterase enzyme (Est5S) and HPLC results revealed that CP could be efficiently degraded up to 100 ppm. Cadusafos (CS), coumaphos (CM), diazinon (DZ) dyfonate (DF), ethoprophos (EP), fenamiphos (FM), methylparathion (MPT), and parathion (PT) were also degraded up to 68, 60, 80, 40, 45, 60, 95, and 100%, respectively, when used as a substrate with Est5S protein. The results highlight the potential use of this enzyme in the cleanup of contaminated insecticides.     

陕西烟蚜茧蜂冬季保种寄主及规模化繁殖的适宜条件研究

在温室中对烟蚜繁育的最适替代寄主展开筛选,测定蚜茧蜂羽化的最适温、湿度,并对烟蚜与僵蚜回接烟苗后的成活率和羽化率进行了测定。结果表明:(1)小白菜繁殖蚜虫量较大,效果优于萝卜和油菜寄主,回接烟苗后烟蚜成活率达85%以上,可作为替代寄主用于烟蚜的冬季保种及规模化繁殖。(2)蚜茧蜂僵蚜羽化的最适温度为20~25℃,最适相对湿度为80%~90%。     

The Adaptation of a Stainless Steel Container for use as a Compression Sprayer

Abstract

Stainless steel sprayers are popular but expensive. The stainless steel tanks manufactured for use with soft drink vending machines can be adapted for use as compression sprayers relatively cheaply. The procedure and materials necessary for adapting the tank are described.     

An improved in vitro rearing system for the human head louse allows the determination of resistance to formulated pediculicides

An improved in vitro rearing system, based on a silicone-reinforced Parafilm^® M membrane, human hair tufts and reconstituted human blood, enabled the large-scale rearing of pediculicide-susceptible (EC-HL) and resistant (SF-HL and BR-HL) strains of human head lice. Developmental time differed for early instars but differences became synchronized as lice matured. Mean survivorship amongst the three strains reared in vitro were not significantly different when compared to EC-HL and SF-HL reared in vivo. The efficacies of three pediculicidal products were assessed under semi-clinical conditions using a modified version of the in vitro rearing system. Treatments of 1% permethrin in acetone, Nix^®, Rid^® or Pronto^® Plus to hair tufts following manufacturer’s instructions were highly efficacious (100% mortality) on EC-HL but differentially efficacious (62.2-84.4% mortality) on SF-HL examined eight days post-treatment. SF-HL that survived the first treatment received an identical second treatment eight days following the first treatment. Survivors (13.3-30.0%) developed to adults (10th-11th day following first treatment) and females successfully laid fertile eggs that developed to first instars. These results confirm resistance to permethrin- and pyrethrin-based pediculicide formulations in SF-HL when assessed under semi-clinical conditions and validates resistance previously determined using filter-paper contact bioassays and unformulated insecticides.     

Allelochemical resistance traits of muskmelon (Cucumis melo) against the fruit fly (Bactrocera cucurbitae) in a hot arid region of India

Host plant resistance is an important component for management of the melon fruit fly, Bactrocera cucurbitae (Coquillett), owing to difficulties associated with its chemical and biological control. Various biochemical traits including total sugar, reducing sugar, non-reducing sugar, tannins, phenols, alkaloids, flavinoid and pH contents of fruit were studied on 11varieties/ genotypes of muskmelon, Cucumis melo L., in relation to resistance against B. cucurbitae under field conditions. Significant differences were found in tested varieties/ genotypes for fruit infestation and larval density per fruit. AHMM/BR-1, RM-50 and AHMM/BR-8 were the most resistant; MHY-5, Durgapura Madhu and Pusa Sarabati were moderately resistant; AHMM/BR-13, Pusa Madhuras and Arka Jeet were susceptible; whereas Arka Rajhans and GMM-3 were the highly susceptible varieties/ genotypes to fruit fly in both seasons, 2011 and 2012. The larval density per fruit increased with an increase in percent fruit infestation and there was a significant positive correlation (r = 0.97) between percent fruit infestation and larval density per fruit. Total sugar, reducing sugar, non-reducing sugar and pH were lowest in resistant and highest in susceptible varieties/ genotypes, whereas tannins, phenols, alkaloids and flavinoid contents were highest in resistant and lowest in susceptible varieties/ genotypes. Total alkaloid and pH contents explained 97.96% of the total variation in fruit fly infestation and 92.83% of the total variation in larval density per fruit due to alkaloids and total sugar contents.     

Construction of a cDNA library of Bemisia tabaci for use in the ''yeast two-hybrid screen'' method

Bemisia tabaci was reported for the first time in the Mediterranean part of Croatia in 2000. It was found in glasshouses in the agricultural area between the towns of Trogir and Omis, on the following crops: Euphorbia pulcherrima , Thunbergia grandiflora , Cucumis sativus (cucumber), Solanum melongena (aubergine), Phaseolus spp. (beans), Ficus carica (fig), Rubus spp. and several weeds of the families Asteraceae and Solanaceae . In 2001, monitoring for the pest was organized all over the country, in each of the 21 counties. In each county, there were several monitoring points so that all the major vegetable and flower producers were included. A special effort was made to record the spread of B. tabaci in the region where it was first found, bearing in mind that optimal conditions for outdoor spread exist along the Adriatic coast. Yellow sticky traps and visual inspection are used to monitor for B. tabaci . Eradication measures are being applied, and regulatory measures have been taken to prevent further spread of B. tabaci to continental parts of Croatia.     

Testing a Rapid Diagnostic Medium for Erwinia amylovora and Development of a Procedure for Sampling Blossoms in Pear Orchards

ABSTRACT The coliform agar produced by Merck was tested for rapid diagnosis of Erwinia amylovora (the causal agent of fire blight) in pear blossoms. The medium enabled the diagnosis to be completed within 36 h. Diagnoses performed with the medium were confirmed by the BIOLOG and the fatty-acid profile methods. The diagnostic medium was used to determine the spatial distribution of colonized blossoms in the orchards and it was found that E. amylovora may be distributed both in clusters and at random. These findings were used in the development of a statistical model for sampling blossoms in the orchard. The model determines the number of trees to be sampled in the orchard and the number of blossoms be taken from each tree, which would enable the true colonization incidence of blossoms in the orchard to be estimated at desired levels of accuracy and confidence. Parameters included in the model are: the total number of trees in the orchard (T), the number of trees to be sampled in the orchard (t), the number of blossoms to be sampled from each tree (n), the true colonization incidence of blossoms (pi), a coefficient of aggregation (rho), the required level of confidence (1 - alpha), and the required level of accuracy (L). Sensitivity analyses revealed that the parameter governing sample size is the required level of accuracy. Sampling of 20 blossoms from each of several hundred trees is required to achieve an accuracy of +/-1%, but only a few single trees are needed for an accuracy level of +/-10%. A sampling procedure then was developed, validated with an independent data set, and found to be accurate. It was concluded that sampling of pear blossoms and estimation of the incidence of blossom colonization by E. amylovora could improve fire blight management, but not in all cases.     

Development of a monoclonal antibody-based immunodetection assay for Botrytis cinerea

Three hybridoma cell lines secreting antibodies that specifically recognize Botrytis cinerea and B.fabae , but not B. allii, have been raised from splenocytes of mice immunized with a low molecular-weight fraction (30 kDa) from surface washings of B. cinerea. Antibodies from these cell lines have been used to develop an antigen-based elisa test that will detect B. cinerea in strawberries. This monoclonal antibody immunoassay detection assay should prove useful to both the cut-flower and wine industries. Supernatants from the three specific cell lines recognize mycelial fragments, saline extracts of mycelia and germinating conidia by both ELISA and immunofluorescence. Recognition of non-germinating spores is poor. Supernatants from the specific cell lines did not recognize other fungi normally involved in post-harvest spoilage of fruits and vegetables. Supernatants from KH4 gave the lowest background values with healthy tissue. Indirect evidence from heat, protease and periodate treatment of the antigens indicates that antibodies from all three specific cell lines are recognizing carbohydrate epitopes on a glycoprotein.     

Development of Strain-specific Primers for a Strain of Gliocladium catenulatum Used in Biological Control

The randomly amplified polymorphic DNA (RAPD) technique was used to develop strain-specific primers for Gliocladium catenulatum strain J1446, which is promising in biological control. One of the primer pairs developed proved to be strain-specific; strain J1446 was differentiated from 16 G. catenulatum strains and six other strains of two Gliocladium species, as well as from Trichoderma virens, and isolates of Nectria spp. and Fusarium spp. Specific primers were also tested with DNA isolated from cucumber leaves, treated or untreated with a solution made from Gliocladium powder. The expected amplification product was produced only from treated leaves. DNA isolated from Gliocladium-treated potato tubers and fungi grown in peat was also used in amplification reactions. Strain-specific primers detected strain J1446 when the amount of DNA was 5pg or more. Some variation between the Gliocladium strains was found by the random amplified microsatellites method (RAMS) and the universally primed polymerase chain reaction method (UP-PCR), but no clear fragments specific to strain J1446 were produced. Cross-blot hybridisation of UP-PCR products differentiated strain J1446 from T. virens, but not from the Gliocladium isolates. The 28S rDNA sequences and -tubulin sequences were identical or very similar in all Gliocladium strains. Thus, it is possible that the Gliocladium strains of the present study are conspecific, which means that a revision in the taxonomy of Gliocladium species may be necessary.     

Gel texture and stability of ascorbic acid and its salts in a calcium alginate rearing medium forSPODOPTERA L1TTORALIS (Boisd.)

The effects of different levels of ascorbic acid, ascorbates and calcium carbonate on the texture of an alginate medium used for rearingSpodoptera littoralis Boisd.. were measured. Diet texture and ascorbic acid stability were found to depend on the Na/Ca ratio in favor of the calcium. The redox potential and vitamin C content of these diets were also examined, showing no significant correlation between these two parameters and age of the media.     

Development of a real-time PCR-based method for detection of Xylophilus ampelinus

A real-time PCR MGB-probe-based detection method specific to Xylophilus ampelinus , the cause of grapevine bacterial blight, was developed. Used in combination with the DNeasy plant mini kit, the sensitivity of X. ampelinus detection was approximately 100 cells from tissue extracts, surpassing the sensitivity of an existing nested PCR method at least tenfold. In field samples a high correlation was observed between real-time PCR cycle threshold (Ct) values obtained and X. ampelinus isolation on artificial media. Isolation was successful from samples with Ct values below 25. Lower concentrations of X. ampelinus , with Ct values up to 36, could also be reliably detected in real-time PCR. The newly developed method offers a reliable and sensitive test for X. ampelinus, suitable as a screening test, complementary to isolation on media or other methods, and could also be used for fast and specific identification of isolated colonies and for relative quantification of X. ampelinus bacteria.     

The use of fomesafen for pre-emergence weed control in cotton

The herbicide fomesafen was found to be selective in preplanting and pre-emergence treatments in cotton (Gossypium hirsutum L.). It was effective due to residual soil activity in controlling some of the most troublesome weeds in cotton fields,i.e., pigweed (Amaranthus spp.), black nightshade (Solarium nigrum L.), velvetleaf (Abutilon theophrasti Medik.) and cocklebur (Xanthium spp.). The best soil activity of fomesafen was achieved from pre-emergence or preplanting applications which were activated when the soil was wetted by rain or sprinkler irrigation, but the herbicide caused damage to the crop’s foliage if rain fell just after the cotton emergence. The most effective and safest method for applying fomesafen in cotton fields was preplanting followed by mechanical incorporation to a depth of 10 cm. Combinations of fomesafen with trifluralin were effective and completed the spectrum of controlled weeds in cotton, including annual grasses, common purslane (Portulaca oleracea L.) and field bindweed (Convolvulus arvensis L.).     

Development of a polyprobe for the simultaneous detection of four grapevine viroids in grapevine plants

This study aimed to develop a polyprobe for the simultaneous detection of four viroids that infect grapevine: Hop stunt viroid (HSVd), Australian grapevine viroid (AGVd), Grapevine yellow speckle viroid-1 and 2 (GYSVd-1, 2), using a non-isotopic dot blot hybridization technique. A polyprobe was constructed by cloning tandem full-length sequences of HSVd, AGVd and GYSVd-1 into a single vector. The cRNA polyprobe detected all four viroids with similar sensitivity to that obtained using individual probes. In addition, samples of 78 varieties from Beijing and Xinjiang were analyzed using the polyprobe to survey the incidence of grapevine viroids in China. The result demonstrated that grapevine viroids were detected in 56 (71.8%) varieties. In this study, a rapid, reliable and cost-effective approach to the simultaneous detection of four grapevine viroids has been developed which has the potential for routine use in quarantine and certification programs.     

Development and evaluation of a model for management of brown rot in organic apple orchards

Temporal development of brown rot (Monilinia fructigena) on fruits was analysed in two organic apple orchards on three apple cultivars in Eastern Hungary from 2002 to 2006. The three-parameter logistic function gave the best fit to brown rot over four non-linear growth functions in all cultivars, years and orchards. Depending on location, year and cultivar, disease increased continuously from 6 to 8 weeks before harvest up to harvest, reaching 19–37% of disease incidence. Disease variables of Y _f , the final disease incidence; β, relative rate of disease progress; AUDPC _S , standardized area under disease progress curve; T _1.5 , the time when disease incidence reaches 1.5% (day), and M, the inflection point were derived from the three-parameter logistic function. The disease variables of Y _f , β, and AUDPC _S were used in a computer simulation for predicting temporal brown rot development, and the disease variables of T _1.5 , M, and Y _f were used to determine threshold values for epidemic intensity. Afterwards these were used to construct a fundamental model for developing a brown rot forecasting and management strategy (BRFMS). The fundamental model contained four parts: i) data insertion and analyses by computer simulation of pathogen submodels, ii) calculation of yield loss threshold levels based on disease incidence, iii) determination of epidemic intensity levels and iv) a decision module with suggestions for disease management practices for each epidemic intensity level. The fundamental model was supplemented with the prediction of occurrence of the first fruit rot symptoms and with the insect injury prediction related to brown rot development in order to complete a BRFMS for organic apple orchards. In a 3-year field evaluation from 2006 to 2008, season-long application of BRFMS treatments reduced the number of sprays against brown rot by 22–33% compared with the treatments of general spray schedules against brown rot.     

Development of a multiplex PCR for the identification of Fusarium solani and F. oxysporum in a single step

Journal of Plant Diseases and Protection - Fusarium solani and F. oxysporum are plant pathogenic fungi that cause root rot and wilt, respectively, in many economically important crops....     

Development of a spot plow providing complete inversion for effective weed control

Tillage for the "complete inversion" of soil, that is, overturning soil slices 180° was proposed, a "spot plow" was developed and tested to accomplish the task, and a simulation model was evaluated to demonstrate the efficacy of the plow on weed control. A 360 mm wide spot plow was designed to operate at a speed of 1.9 m s⁻¹ for the spot plowing with the least possible lateral displacement of the soil slice by utilizing the inertia of the soil slice and securely rotating it. In field experiments, complete spot inversion required an operating speed of at least 1.6 m s⁻¹; at lower speeds, a portion of the soil block was left half-inverted and further lowering led to considerable lateral displacement. The displacement in the forward and lateral directions was minimal, implying that spot plowing is suitable for potential application to and verification of the weed population dynamics model in the field. A simple linear matrix model of the population dynamics of annual weeds was proposed, whereby four layers of soil were set to describe tillage and other ecological events. The effect of tillage on weed control was evaluated by the equilibrium reproduction rate allowed to sustain a stable population of weeds. The simulation model showed that alternately changing the depth of spot plowing had a significant effect on controlling weeds of low-survival-rate seeds, even when some incomplete inversion of the soil slice was taken into account.     

Development of a polyclonal antibody-based immunoassay for the early detection of Sclerotinia sclerotiorum in rapeseed petals

A serological test has been developed that allows the early detection of infection of young petals by Sclerotinia sclerotiorum , an important pathogen of rapeseed. Two steps were required to obtain an antiserum sufficiently specific for S. sclerotiorum. Soluble mycelial extracts of S. sclerotiorum were used to produce the first generation polyclonal antiserum. This was not specific for S. sclerotiorum in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and allowed the screening of cross-reacting fungal species such as Botrytis cinerea , a pathogen commonly present on rapeseed petals. Using a polyclonal anti- B. cinerea serum enabled the absorption, by serial cycles, of S. sclerotiorum antigens common to B. cinerea. Residual antigens were then used as immunogens for the production of two second generation antisera (S1 and S2) which were then tested by DAS-ELISA. Cross-reactions with B. cinerea decreased with purification cycles of the immunogen whereas Cross-reactions with some unrelated fungi slightly increased. S. sclerotiorum and B. cinerea were distinguishable using antiserum S2.     

Development of a monoclonal antibody immunoassay for the eyespot pathogen Pseudocercosporella herpotrichoides

A double-antibody-sandwich ELISA test has been developed for the detection of Pseudocercosporella herpotrichoides using a highly specific monoclonal antibody PH-10 as the capture antibody and genus-specific rabbit polyclonal antiserum as the detector antibody. The assay recognizes extracts from plants both artificially and naturally infected with P. herpotrichoides giving at least three-fold higher absorbance values with extracts from Pseudocercosporella-infected tissue than with extracts from healthy tissues or from tissues naturally infected with Microdochium nivale, Rhizoctonia cerealis or material artificially inoculated with P. anguioides. The assay tested positively against all isolates of P. herpotrichoides , including both W-type and R-type isolates. In this assay system, extraction of the antigen from the stem bases of infected plants is a one-step process not requiring any dilution procedures. The assay can be used to detect the pathogen in presymptomatic infected seedlings. The immunogen used to generate the specific monoclonal antibody and the rabbit antiserum was a mycelial extract from which the high-molecular-weight proteins and glycoproteins had been removed by ammonium sulphate precipitation. The high-molecular-weight fraction was shown to contain cross-reactive antigens; it induced antiserum in mice that cross-reacted with the other stem-base fungi even at high dilutions. The monoclonal antibody PH-10 is an IgM antibody. Heat and periodate treatment of the antigen indicate that it is a glycoprotein and that the epitope recognized by the antibody is a protein.