In vitro production of the biological control agent Heterorhabditis indica SL0708 in different agar media

ABSTRACT

Heterorhabditis indica SL0708 is an entomopathogenic nematode isolated from Valle del Cauca-Colombia, whose bacterial symbiont, Photorhabdus luminescens subsp. akhurstii SL0708, has potential to control pests of economic importance in Colombia. Since in vivo production does not supply its demand, this investigation evaluated H. indica SL0708 production on different agar media. Five culture media (I, II, III, IV and V) were evaluated for productivity and pathogenicity of infective juveniles (IJs). IJs emerged between 11 and 16 days after inoculation in all media, with a total of 2.7?×?10⁴ and 4.7?×?10⁶ IJs produced during 15 days after IJs emergence, with maximum productivity at day five and high variability. Pathogenicity to Galleria mellonella larvae was not significantly different between in vitro and in vivo produced IJs on all media tested. Media IV and V were selected for their higher productivity. Subsequently, nematode inoculum size was evaluated in selected media at 2000, 4000 and 6000?IJs ml^?1, but significant differences were not observed in productivity and pathogenicity. Lastly, lipid source influence was evaluated in medium IV comparing canola, olive and soy oils. None of the plant-based oils had a significant effect on IJs production and pathogenicity. A medium was selected for H. indica SL0708 IJs production which was suitable in terms of productivity, culture time and pathogenicity of IJs produced. The medium and parameters selected in this study could be applied as an alternative for mass production of this entomopathogenic nematodes.     

In vitro solid-state production of Steinernema innovationi with cost analysis

Low-cost mass production of entomopathogenic nematodes (EPNs) is an important prerequisite towards their successful commercialisation. This study evaluated six low-cost solid substrate media for in vitro mass production of Steinernema innovationi. Cost analysis was undertaken and an estimated retail price was calculated. This was then compared to the costs of commercial EPN products currently on the market. The highest yield of infective juveniles (IJs) was obtained from a medium containing a puree of house fly, Musca domestica, larvae?+?0.15?g canola oil, (781,678?±?221 IJs/5?g medium). This medium also had the lowest number of adults remaining in the medium and dead IJs (<10%) at the time of harvest (Day 28). The estimated retail price (R243.27 per 50 million IJs) for S. innovationi produced with our solid culture system was considerably lower than the market price for other Steinernema species products sold by E～nema, BASF corporation, Koppert, BioBest and Natural Insect Control. The production system developed in this study offers a competitive technology to produce EPN products without having to invest in large-scale liquid fermentation equipment, by using a relatively cheap production medium and simple solid culture growing conditions using Erlenmeyer flasks.     

Infectivity of Steinernema mushtaqi (Rhabditida: Steinernematidae) against insect pests and their mass production

The infectivity of entomopathogenic nematode (EPN), Steinernema mushtaqi was tested against legume pod borer, Maruca vitrata, tobacco caterpillar, Spodoptera litura, blue butterfly, Lampides boeticus, red hairy caterpillar, Amsacta moorei, brown bug, Clavigralla gibbosa, mealy bug, Centrococcus somatics, fruit borer, Earias vittella and green bug, Nezara viridula larvae and in vivo mass production of the above-tested species of EPN have been carried out during 2008. S. mushtaqi was found to be more pathogenic to A. moorei, as it brought about 100% mortality within 48 h, than to S. litura, L. boeticus, N. viriduala and E. vittella, as mortality occurred within 72 h; whereas this level of mortality was recorded in C. somatis, C. gibbosa and M. vitrata within 144 h. No mortality was observed in the control treatment. Multiplication of S. mushtaqi and the yield of infective juveniles (IJs) on these insects was the highest (0.94 × 10⁵ IJs/cadaver) from N. viriduala, followed by S. litura (0.76 × 10⁵ IJs/cadaver), L. boeticus as also C. gibbosa (0.31 × 10⁵ IJs/cadaver) and M. vitrata (0.20 × 10⁵ IJs/cadaver). Very poor populations of IJs were found from A. moorei (0.15 × 10⁵ IJs/cadaver) and C. somatics (0.01 × 10⁵ IJs/cadaver). No multiplication of IJs was found from the cadaver of E. vittella. This opens a new hope of utilising S. mushtaqi in the insect pests management programme.     

Mechanical production of pellets for the application of entomopathogenic nematodes: effect of pre-acclimation of Steinernema glaseri on its survival time and infectivity against Phyllophaga vetula

The low survival time and diminished infectivity by entomopathogenic nematodes (EPNs) from granular formulations limit their efficiency against agricultural insect pests. This study determined the benefit of pre-acclimating infective juveniles (IJs) of Steinernema glaseri (NJ-43 strain) on extending their mean survival time (ST_m) in diatomaceous earth (DE) pellets and increasing their infectivity against Phyllophaga vetula. The IJs were reared in Galleria mellonella larvae placed in Petri dishes containing plaster of Paris (PP) or modified White traps (WTs). Pelletisation was performed in a machine operating on the principle of laminar flow using DE Celite® 209. Pellets were stored at room temperature (23?±?3°C) and high relative humidity (96–100%). IJs harvested from WTs between the 3rd and the 5th days after the onset of emergence were more infective on P. vetula and pre-acclimation of S. glaseri in PP increased significantly its ST_m in the pellets; from 23.1 to 34.5 days, compared with non-pre-acclimatised IJs from WTs. However, juveniles with or without pre-acclimation formulated in DE pellets failed to achieve significant control of P. vetula. These results are discussed in light of the relationship between EPN survival and host infection by EPNs with possible effects of the formulation in DE pellets.     

Storage of osmotically treated entomopathogenic nematode Steinernema carpocapsae

The infective juveniles (IJs) of Steinernema carpocapsae‘All’ were osmotically stressed by a mixture of ionic (fortified artificial seawater) and non‐ionic (3.2 mol/L glycerol) solutions to establish a method for osmotic storage of entomopathogenic nematodes. Seven combinations (termed solution A to G) with different proportions of these two solutions were tested, with sterile extra pure water (sepH₂O, termed solution H) as a control. The mortality of the IJs at a concentration of 5 × 10⁵ IJ/mL in the solutions A to G, and H were 13.2%, 16.2%, 16.7%, 13.5%, 25.2%, 31.6%, 44.6%, and 1.0%, respectively, after 21 days storage at 25°C. Most of the IJs shrunk and stopped motility after 6–9 hours incubation at 25°C in solutions A to D. Based on the results, solutions A to D and H were chosen to further test the osmotic survival of the IJs at different IJ concentrations (5 × 10⁵, 2.5 × 10⁵, 2 000 IJ/mL) and incubation temperature (30°C, 25°C, 10°C). The resulting IJs were exposed to a high temperature assay (45°C for 4 h, HTA). Osmotically stressed IJs showed improved heat tolerance. The mortality of the IJs increased with the increasing concentrations of the test IJs and the storage temperatures after exposing to the HTA. More than 88.4%, 62.3% or 2.4% of the treated IJs died at the above three IJ concentrations, respectively. At the three IJ concentrations (2 000 IJs/mL, 2.5 × 10⁵ IJs/mL or 5 × 10⁵ IJs/mL), the highest mortality was recorded in solution D (11.6%, 85.9% or 98.0%, respectively), and the lowest mortality in solution B (2. 4%, 62.3% or 86.6%, respectively). No untreated IJs survived after the heat treatment. During 42 days storage at 10°C, the IJs mortality in the solutions A to D and H were 7.19%, 5.97%, 4.41%, 4.34%, and 4.34% respectively, and showed no significant differences. In conclusion, osmotic treatment of the IJs of S. carpocapsae‘All’ in a mixture of ionic and non‐ionic solutions enhances the heat tolerance. The mortality of the IJs after HTA increased with the increasing concentrations of the test IJs and the storage temperatures after exposure to the HTA. The result is promising for the osmotic storage of the entomopathogenic nematodes.     

Production ofBeauveria bassiana [Deuteromycotina: Hyphomycetes] in different liquid media and subsequent conidiation of dry mycelium

Mycelium ofBeauveria bassiana can be grown in liquid culture, filtered, and the mycelium dried. After rehydration the mycelium sporulates. Two carbohydrate sources (sucrose and maltose), and one nitrogen/vitamin source (yeast extract) were tested for mycelium growth and subsequent conidial production. Maximum mycelium growth (12.31 mg/ml), in liquid culture, was in the sucrose (3.5%)/yeast extract (3.5%) medium, but mycelium from a maltose (2%)/yeast extract (0.75%) medium produced the maximum of 4.62×10⁶ conidia/mg dry mycelium after incubation in moist Petri dishes. Using the data on mycelium yield (in liquid culture) and conidial production (by dry mycelium) it is calculated that the sucrose (3.5%)/yeast extract (3.5%) and the maltose (2%)/yeast extract (0.75%) media produce most conidia per media volume (an equivalent of 3.52–3.72×10⁷ conidia/ml).      

Design of an efficient fermentation process for the production of Metarhizium acridum blastospores

Using a sequential approach, we described efficient blastospore production in a stirred tank bioreactor (3?L capacity). We used the response surface methodology to optimise the media ingredients and fermentation parameters to obtain the maximum production of blastospores by a locally collected isolate of Metarhizium acridum (Ascomycota: Hypocreales). The results showed that a liquid culture medium supplemented with monopotassium phosphate (15.17?g/L), corn steep liquor (69.25?g/L), and casamino acids (80.68?g/L) in a stirred tank bioreactor under operating conditions constant at 635?rpm, a temperature of 26°C, and pH 3.3 produced 1.25?×?10⁸?blastospores (bls)/ml, with 93% viability after 120?h of fermentation. This bioreactor yield compares favourably with the yields obtained by shake flask production and confirms the suitability of the media and production parameters for the potential scale-up fermentation production of M. acridum.     

Growth and Immunologic Studies on the Culture Forms of Trypanosoma lewisi*

SYNOPSIS. Culture forms of Trypanosoma lewisi grown at 27 C in a diphasic blood agar medium resemble in structure the stage found in the invertebrate host. Cultures inoculated with approximately 1 × 10⁶ trypanosomes/ml attain maximum populations of 2–7 × 10⁷ organisms/ml after 5–6 days of incubation. The stationary phase persists 6–15 days. The decline of the population is of relatively long duration with approximately 1 × 10⁶ viable organisms/ml present after 90 days. Variations in growth were attributed to the preparation of defibrinated heated rabbit blood incorporated into the culture medium. With inocula of 3.0 × 10⁵ trypanosomes/ml there was a lag in growth not observed with larger inocula. Trypanosomes incubated at elevated temperatures had altered growth curves compared to organisms at 27 C. Agitation of cultures did not affect the growth or stationary phases, but hastened the population decline. Heated and unheated 5% (v/v) normal rat serum incorporated in the liquid phase of the medium altered the growth of the organisms. Heated serum caused a decrease in the population and an extended lag phase. The effects on growth were more marked with unheated serum suggesting that both heat-stable and labile components affect growth. Antisera from rats injected with live culture forms included in the liquid phase inhibited, while antisera from rats 24 days after infection with the blood stream forms had no effect on the growth of the culture forms. Antisera from rabbits immunized with sonicates of culture forms also altered the growth of the organisms in culture. Rabbit antisera prepared by immunization with sonicates of dividing and non-dividing blood stream forms had no effect on the in vitro growth. Antisera from animals immunized with rat blood and culture medium were also without effect. The immunologic implications of the data are considered and discussed.     

Effect of insect cadaver desiccation and soil water potential during rehydration on entomopathogenic nematode (Rhabditida: Steinernematidae and Heterorhabditidae) production and virulence

We examined the influence of insect cadaver desiccation on the virulence and production of entomopathogenic nematodes (EPNs), common natural enemies of many soil-dwelling insects. EPNs are often used in biological control, and we investigated the feasibility of applying EPNs within desiccated insect cadavers. Desiccation studies were conducted using the factitious host, Galleria mellonella (Lepidoptera: Pyralidae, wax moth larvae) and three EPN species (Heterorhabditis bacteriophora ‘HB1’, Steinernema carpocapsae ‘All’, and Steinernema riobrave). Weights of individual insect cadavers were tracked daily during the desiccation process, and cohorts were placed into emergence traps when average mass losses reached 50%, 60%, and 70% levels. We tracked the proportion of insect cadavers producing infective juveniles (IJs), the number and virulence of IJs produced from desiccated insect cadavers, and the influence of soil water potentials on IJ production of desiccated insect cadavers. We observed apparent differences in the desiccation rate of the insect cadavers among the three species, as well as apparent differences among the three species in both the proportion of insect cadavers producing IJs and IJ production per insect cadaver. Exposure of desiccated insect cadavers to water potentials greater than −2.75 kPa stimulated IJ emergence. Among the nematode species examined, H. bacteriophora exhibited lower proportions of desiccated insect cadavers producing IJs than the other two species. Desiccation significantly reduced the number of IJs produced from insect cadavers. At the 60% mass loss level, however, desiccated insect cadavers from each of the three species successfully produced IJs when exposed to moist sand, suggesting that insect cadaver desiccation may be a useful approach for biological control of soil insect pests.     

Production ofbeauveria bassiana [Deuteromycotina. Hyphomycetes] sympoduloconidia in submerged culture

Submerged conidiation of the entomogenous HyphomyceteBeauveria bassiana is reported. Conidiogenous cells produce sympoduloconidia on conidiogenous cells in liquid shaker culture; hyphal bodies and mycelium fragments are also produced. The morphology of these fungal structures is discussed and illustrated. Several simple liquid media are tested for the production of conidia and hyphal bodies. Maximum yields of conidia (170×10⁶ conidia/ml) are produced in a medium consisting of sucrose (2%) — yeast extract (0.5%) and basal salts, and maximum yields of hyphal bodies (740×10⁶ hyphal bodies/ml) in a sucrose (2.5%) — yeast extract (2.5%) medium.      

Monoxenic liquid culture with Escherichia coli of the free-living nematode Panagrolaimus sp. (strain NFS 24-5), a potential live food candidate for marine fish and shrimp larvae

The free-living, bacterial-feeding nematode Panagrolaimus sp. (strain NFS 24-5) has potential for use as live food for marine shrimp and fish larvae. Mass production in liquid culture is a prerequisite for its commercial exploitation. Panagrolaimus sp. was propagated in monoxenic liquid culture on Escherichia coli and parameters, like nematode density, population dynamics and biomass were recorded and compared with life history table data. A mean maximum nematode density of 174,278 mL^?1 and a maximum of 251,000 mL^?1 were recorded on day 17 after inoculation. Highest average biomass was 40 g L^?1 at day 13. The comparison with life history table data indicated that the hypothetical potential of liquid culture is much higher than documented during this investigation. Nematode development is delayed in liquid culture and egg production per female is more than five times lower than reported from life history trait analysis. The latter assessed a nematode generation time of 7.1 days, whereas the process time at maximum nematode density in liquid culture was 16 days indicating that a reduction of the process time can be achieved by further investigating the influence of nematode inoculum density on population development. The results challenge future research to reduce process time and variability and improve population dynamics also during scale-up of the liquid culture process.     

The reproduction potentials of four entomopathogenic nematode strains related to cost-effective production for biological control

Bioassays to evaluate the mortality, virulence and reproduction potentials of four indigenous EPN strains, S-PQ16, S-BM12, H-KT3987 and H-CB3452 on insect larvae of mealworm (Tenebrio molitor) and greater wax moth (Galleria mellonella) revealed the highest mortality rates of two insect larvae at the highest inoculation dose of 100 IJs to range from 89 to 100 percent and 94.3–100 percent at 48 h after inoculation, respectively. Virulence was high for all nematode strains, with LC₅₀ values between 29.6 and 47.3 IJs/insect host. The highest IJ yields were different between nematode strains and insect host, from 66.8 × 10³ IJs (S-PQ16) to 118.6 × 10³ IJs (H-KT3987) on T. molitor, and from 54.2 × 10³ IJs (S-BM12) to 163.3 × 10³ IJs (H-KT3987) on G. mellonella. The culturing cost in terms of food expenditure for rearing insect larvae varied between insect larvae and nematode strains, from 6.76 to 26.63 USD per billion IJs for nematode strains cultured on T. molitor larvae and from 3.54 to 7.81 USD per billion IJs for nematode strains cultured on G. mellonella larvae. The full cost for a nematode product of 2.5 × 10⁹ IJs per hectare, produced through in vivo mass culturing, of the most efficient nematode strain, H-KT3987, was 191.3 USD, slightly cheaper than 199.4 USD for the same nematode product produced through in vitro mass culturing.     

Laboratory and semi-field evaluation of three entomopathogenic nematode species on two non-target insect predators,Coccinella septumpunctata (Coleoptera: Coccinellidae) and Chrysoperla carnea (Neuroptera: Chrysopidae)

Abstract

Biocontrol potential of the entomopathogenic nematodes (EPNs) on the second-instar larvae of the non-target insect predators, Coccinella septumpunctata and Chrysoperla carnea as compared to Spodoptera littoralis (Boisd.) was evaluated. The pathogenicity of EPNs, namely, Heterorhabditis bacteriophora, Steinernema feltiae and Steinernema carpocapsae at concentrations 100, 200, 400, 800 and 1600 IJs/cup) were tested at 2, 4 and 6 days’ post-inoculation. Laboratory results showed significant differences among the mortality rates of different tested larvae, for each concentration at different time intervals. H. bacteriophora induced the highest mortality followed by S. carpocapsae treatment. However, S. feltiae was found to be more safety on predators as it causes less mortality at 6 days of treatment. The values of half lethal concentrations (LC₅₀) were 614.06, 3797.43 and 676.47 IJs/cup for C. Carnea and 390.60, 1209.88 and 503.65 IJs/cup for C. septumpunctata treated by H. bacteriophora, S. feltiae and S. carpocapsae, respectively. In semi-field experiments, there were non-significant differences among mortality of each predator indicated at concentrations of the different EPNs after 2 days or 6 days’ post-inoculation. The study revealed a lethal pathogenic effect of EPNs against insect pests but caused low mortality on the non-target ones.     

Behavioural and physiological responses of infective juveniles of the entomopathogenic nematode Heterorhabditis to desiccation

Infective juveniles (IJs) of entomopathogenic nematodes (EPNs) are susceptible to a wide variety of environmental factors, including desiccation, which limit their usefulness as biocontrol agents. Although EPNs can be subjected to a gradual loss of water in their natural environment they are not full anhydrobiotes, being able to survive only moderate levels of desiccation at high relative humidities (rh). We investigated the desiccation tolerance of IJs of several Heterorhabditisspecies and strains when exposed to fast and slow desiccation regimes. We also investigated the behavioural and biochemical responses of Heterorhabditis IJs when exposed to 98% rh for 4 days. IJs of H. megidis UK211 (but not IJs of H. indica) aggregate into large clumps when desiccated at high rh, but unlike Steinernema spp., neither H. megidis nor H. indica IJs showed any tendency to coil. Preincubation of H. megidis UK211 IJs at high (98%) rh enhances their ability to survive for 150 min at 57% rh. We show that preincubation of H. megidis and H. indica at 98% rh induces the synthesis of glycerol but not of trehalose, whereas identical preincubation conditions do induce trehalose synthesis in Steinernema carpocapsae and Aphelenchus avenae. The biosynthesis of glycerol rather than trehalose by IJs of two species of Heterorhabditis in response to moderate levels of desiccation indicates that Heterorhabditis is unlikely to have the necessary metabolic responses to desiccation required to enable it to enter into a fully anhydrobiotic state.     

Efficacy of entomopathogenic nematodes against potato tuber moth,Phthorimaea operculella (Lepidoptera: Gelechiidae) under laboratory conditions

The susceptibility of potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) to native and commercial strains of entomopathogenic nematodes (EPNs) was studied under laboratory conditions. Native strains of EPNs were collected from northeastern Iran and characterised as Steinernema feltiae and Heterorhabditis bacteriophora (FUM 7) using classic methods as well as analysis of internal transcribed spacer (ITS) and D2/D3 sequences of 28S genes. Plate assays were performed to evaluate the efficiency of five EPN strains belonging to four species including Steinernema carpocapsae (commercial strain), S. feltiae, Steinernem glaseri and H. bacteriophora (FUM 7 and commercial strains). This initial assessment with 0, 75, 150, 250, 375 and 500 IJs/ml concentrations showed that S. carpocapsae and H. bacteriophora caused the highest mortality in both larval and prepupal stages of P. operculella, PTM. Thereafter, these three strains (i.e. S. carpocapsae, H. bacteriophora FUM 7 and the commercial strains) were selected for complementary assays to determine the effects of soil type (loamy, loamy–sandy and sandy) on the virulence of EPNs against the second (L2) and fourth instar (L4) larvae as well as prepupa. A soil column assay was conducted using 500 and 2000 IJs in 2-ml distilled water. Mortality in the L2 larvae was not affected by the EPN strain or soil type, while there was a significant interactive effect of nematode strains and soil type on larval mortality. The results also showed that EPN strains have higher efficiency in lighter soils and caused higher mortality on early larvae than that in loamy soil. In L4 larvae, mortality of PTM was significantly influenced by nematode strain and applied concentrations of infective juveniles. The larval mortality induced by S. carpocapsae was higher than those caused either by a commercial or the FUM 7 strain of H. bacteriophora. Prepupa were the most susceptible stage.     

Production of Trichoderma micropropagules as a biocontrol agent in static liquid culture conditions by using an integrated bioreactor system

ABSTRACT

In this study, we optimised the conditions for the production of micropropagules of Trichoderma harzianum EGE-K38 in static liquid culture in Modified Czapec Medium (MCM) containing 8?g/L glucose in an integrated tray bioreactor system designed by our research group. Incubation temperature, air flow rate, inoculum spore concentration, inoculation size, medium volume and the use of spores or agar plugs containing mycelia as inoculum were individually studied as one factor at a time. The maximum micropropagule count was 5.2?±?0.2?×?10^9?cfu/mL and dry cell weight was 17?±?2?g/L. For the subsequent drying processes, the maximum drying yield percentage ((viable micropropagule counts after drying/viable cells before drying)*100) after drying of micropropagules was 23.30% (cfu/cfu). Results obtained from our integrated tray bioreactor system showed that static liquid culture fermentation offers potential for industrial scale fungal BCAs production.     

Mass cultivation and storage of the rhabditid nematode Phasmarhabditis hermaphrodita,a biocontrol agent for slugs

The rhabditid nematode Phasmarhabditis hermaphrodita (a parasite capable of killing pest slugs) was grown in vitro, in association with a mixed bacterial flora on foam chips impregnated with a kidney‐based nutrient medium in aerated bags, to provide sufficient numbers for laboratory and field experiments. The feasibility of producing nematodes in liquid culture was investigated using 250 ml flasks. Baffled flasks containing 25 or 50 ml or liquid were found to be better than baffled flasks containing 100 ml or unbaffled flasks. Inoculum densities ranging from 50 to 330 ml^‐1 did not affect final yield. Dauer larvae in aerated water died rapidly at temperatures of 26–35°C. Survival was progressively better at 22°C and 15°C, and best at 5°C or 10°C.     

Combined releases of entomopathogenic nematodes and the predatory mite Hypoaspis aculeifer to control soil-dwelling stages of western flower thrips Frankliniella occidentalis

The effect of the predatory miteHypoaspis aculeifer Canestrini (Acarina:Laelapidae) on soil-dwelling stages of thewestern flower thrips (WFT) Frankliniellaoccidentalis Pergande (Thysanoptera: Thripidae)and the influence of combined releases of H.aculeifer and two entomopathogenic nematodes(EPNs) Heterorhabditis bacteriophora Poinar(Rhabditida: Heterorhabditidae) (strain HK3,HK3) and Steinernema feltiae Filipjev(Rhabditida: Steinernematidae) (Nemaplus®,SFN) were investigated in pot trials usingseedlings of green beans (Phaseolus vulgarisL.). Ten H. aculeifer adults per pot and 400infective juveniles (IJs) cm^–2 soil, of the twoEPN strains were used. In comparison withuntreated control, H. aculeifer reduced theproportion of adult F. occidentalis emergenceby 46%, while SFN and HK3 led to a reductionin adult thrips emergence by 46% and 61%,respectively. Significant differences in adultWFT emergence were found between combinedtreatments of EPNs and H. aculeifer, andindividual applications of EPNs and/or H.aculeifer, with significantly lower adultthrips emergence in the combined treatments.These findings highlight the potential for acombined use of EPNs with H. aculeifer for thecontrol of soil-dwelling stages of thrips.     

Development of a quantitative method for determination of the optimal conditions for protoplast isolation from cultured plant cells

An index [kv: average isolation rate of viable protoplast (number/ml min)] was established to evaluate the optimal conditions for protoplast isolation from cultured plant cells. The optimal conditions for protoplasts isolation from Nicotiana tabacum BY2 cultured cells could be determined on the basis of the kv [31.7 × 10³ (number/ml min)]. The colony-forming efficiency of the protoplasts was about 46%. The optimal conditions for protoplasts isolation from Catharanthus roseus [kv = 38.1 × 10³ (number/ml min)] and Wasabia japonica [kv = 14.2 × 10³ (number/ml min)] cultured cells could also be determined. Furthermore, a method for rapid regenerating cell wall of protoplast in liquid culture using alginate gel containing locust bean gum was developed.     

Post-application of anti-desiccant agents improves efficacy of entomopathogenic nematodes in formulated host cadavers or aqueous suspension against diapausing codling moth larvae (Lepidoptera: Tortricidae)

Codling moth (CM), Cydia pomonella (L.) is the most serious pest of apple and other pome fruit worldwide. In temperate climates, diapausing cocooned larvae make up 100% of the population. Control of this stage would reduce or eliminate damage by first generation CM in late spring and early summer. Entomopathogenic nematodes (EPNs) are good candidates for control of CM in the cryptic habitats where the larvae overwinter. The two predominant limiting factors for EPNs are adequate moisture and temperatures below 15°C. Formulation that maintains moisture and enables survival of EPN infective juveniles (IJs) until they can infect overwintering larvae would significantly improve their utility for protection of apple, pear and walnut. In laboratory studies conducted in moist mulch (consisting of apple and conifer wood), Galleria mellonella (L.) larvae infected with Steinernema carpocapsae (Weiser), S. feltiae (Filipjev), or Heterorhabditis bacteriophora Poinar and coated with starch and clay, produced mean mortalities of 42, 88, and 24%, respectively in CM larvae. Mulched field plots treated with formulated S. carpocapsae- or S. feltiae-infected G. mellonella larvae, then followed by an application of wood flour foam as an anti-desiccant, resulted in 56 and 86% mortality, respectively. Comparative tests of aqueous suspensions of S. carpocapsae IJs applied to cardboard bands on apple tree trunks followed by water, fire retardant gel or foam resulted in 11, 35, and 85% respective mortalities. Identical tests with S. feltiae resulted in 20, 19, and 97% respective mortalities. Our research with cadaver formulations of EPNs in mulch and aqueous suspensions on tree trunks combined with anti-desiccant agents, demonstrated significant improvement in larvicidal activity for diapausing cocooned CM larvae.