A new oil-based formulation of Trichoderma asperellum for the biological control of cacao black pod disease caused by Phytophthora megakarya

In African cacao-producing countries, control of cacao black pod disease caused by Phytophthora megakarya is a priority. Introducing biological control agents as part of a P. megakarya control strategy is highly desirable, especially in a perspective of pesticide reduction. Trichoderma species are among the most used biological control agents. In Cameroon, Trichoderma asperellum formulated in wettable powder has produced positive effects against this disease. However, with this type of formulation, shelf-life and persistence of conidia on pods are limited. Our study therefore sought to develop a new T. asperellum formulation that would be more effective and better suited to the conditions of field application by small-scale producers in Cameroon. We selected a soybean oil-based oil dispersion, in which the half-life of the conidia reached 22.5 weeks, versus 5 weeks in aqueous suspension. Tested on detached pods, the formulation completely inhibited the development of the disease. When sprayed in the field on cacao clones highly sensitive to P. megakarya, the formulation resulted in 90% protection of treated pods after 1 week, and 50% after 3.2 weeks. The formulations exercised a measurable effect for up to 7 weeks, versus 2 weeks in the case of an aqueous conidial suspension and 5 weeks for that of a conventional fungicide (Kocide). Trichoderma asperellum formulated in oil dispersion has therefore great potential for the control of cacao black pod disease with less recourse to synthetic fungicides. Moreover, this formulation is well adapted to the types of sprayers used by small-scale cacao producers in Cameroon.     

Cellular damage during drying and storage of Trichoderma harzianum spores

Factors that cause cellular damage during the drying and storage of Trichoderma harzianum conidia were independently studied to determine their effects on spore viability. Specifically, thermal stress and dehydration levels (water activity, a_w = 0.1–0.7) were assessed for their effect on spore survival. In addition, environmental conditions, such as water activity and temperature, were evaluated during storage of the spores. T. harzianum spores produced in liquid culture are highly sensitive to thermal stress, but dehydration does not seem to be a factor that influences spore death during desiccation. An inverse correlation between spore survival and the specific concentration of malondialdehyde (MDA) was observed during storage, especially when the conidia moisture levels were lower than the monolayer moisture levels. We prepared spore suspensions without additives and spray-dried the samples. Our data showed that reduced sample viability was mainly caused by the temperature of the drying process, an effect that appears to be independent of water activity.     

Microencapsulation of Bacillus subtilis B99-2 and its biocontrol efficiency against Rhizoctonia solani in tomato

Biological control is a promising approach to protecting plants from disease. Bacillus subtilis has been widely used in agriculture for promoting plant growth and biocontrol. However, their short shelf life limits the application of biological pesticides. The objectives of this study were to develop a microencapsulation procedure of B. subtilis B99-2 using maltodextrin and gum arabic as wall materials to determine the optimum conditions of spray-drying in microencapsulation, evaluate storage stability of microcapsules, and assess their biocontrol efficiency against Rhizoctonia solani in tomato under field conditions. We microencapsulated the Bacillus thallus by spray-drying with various concentrations of the wall material. Maltodextrin was found to be an efficient wall material, especially at concentrations higher than 80%, while gum arabic did not affect the bacterial survival rate. The mean survival rate of B. subtilis was more than 90%, when spray drying was performed at 145 °C, with a feed flow rate of 550 mL h⁻¹, and a spray pressure of 0.15 MPa. B. subtilis microcapsule survival rate was 87.53% after 540 d of storage, which was a longer shelf life than that of wettable powders. Moreover, its biocontrol efficacy reached 79.91% when a dosage of 300 g hm⁻² was used, the microcapsule showed higher control efficacy than Thiram wettable powder against R. solani in tomato under field conditions. All these characteristics indicated that B. subtilis microcapsules have the potential to become a successful biocontrol product.     

Fungal interaction between Trichoderma spp. and Pleurotus ostreatus on the enriched solid media with licorice Glycyrrhiza glabra root extract

The aim of this study is to investigate the antifungal activity of mycelia of Pleurotus ostreatus (white oyster mushroom) and licorice (Glycyrrhiza glabra) root extract against three undesirable fungi. They are Trichoderma spp., Trichoderma harzianum I and Trichoderma harzianum II which was tested on PSA (potato sucrose agar) medium enriched with licorice (Glycyrrhiza glabra) root extract (PSA-G media) using three concentrations (0.05, 0.10 and 0.20 g/L) in alone and dual cultures. Trichoderma spp. showed less mycelial growth of 8.75, 9.17 and 9.50 mm/day on PSA-G0.05, PSA-G0.1 and PSA-G0.2 respectively compared with 10.25 mm/day on fresh PSA (control) in dual culture. The best mycelial growth inhibition was recorded on PSA-G0.2 (14.97%) by T. harzianum II in alone culture opposite 63.72% in dual ones. The lower mycelial growth rate of T. harzianum I was 17.75 mm/day on PSA-G0.1 (0.10 g/L). In dual culture, overgrowth time of T. harzianum I had 5 days compared as approx. 6 days in alone culture. Generally, when the concentration of licorice extract increased, the mycelial growth rate of the undesirable fungi decreased. Also, all PSA-G media, especially PSA-G0.2, indicated low growth averages compared with the control (fresh PSA) against the pathogen while this concentration encourages growth of oyster mushroom. Also, this concentration reduced the density of sporulation of green molds; therefore, this concentration can be applied to reduce influence this pathogen in cultivation farm.     

Soil microbial biomass influence on growth and biocontrol efficacy of Trichoderma harzianum

Hyphal growth and biocontrol efficacy of Trichoderma harzianum may depend on its interactions with biotic components of the soil environment. Effects of soil microbial biomass on growth and biocontrol efficacy of the green fluorescent protein transformant T. harzianum ThzID1-M3 were investigated using different levels of soil microbial biomass (153, 328, or 517 μg biomass carbon/g of dry soil). Hyphal growth of T. harzianum was significantly inhibited in soil containing 328 or 517 μg biomass carbon/g of dry soil compared with soil containing 153 μg biomass carbon/g. However, when ThzID1-M3 was added to soil as an alginate pellet formulation, recoverable populations of ThzID1-M3 varied, with the highest populations in soil containing 517 μg biomass carbon/g. When sclerotia of Sclerotinia sclerotiorum were added to soils (10 sclerotia per 150 g soil) with ThzID1-M3 (20 pellets per 150 g soil), colonization of sclerotia by ThzID1-M3 was significantly lower in the soil containing the highest level of biomass. Addition of alginate pellets of ThzID1-M3 to soils (10 pellets per 50 g) resulted in increased indigenous microbial populations (total fungi, bacteria, fluorescent Pseudomonas spp., and actinomycetes). Our results suggest that higher levels of microbial soil biomass result in increased interactions between introduced T. harzianum and soil microorganisms, and further that microbial competition in soil favors a shift from hyphal growth to sporulation in T. harzianum, potentially reducing its biocontrol efficacy.     

Management of white mold in processing tomatoes by Trichoderma spp. and chemical fungicides applied by drip irrigation

Field trials were carried out to evaluate six treatments combining biological agents and chemical fungicides applied via chemigation against white mold (Sclerotinia sclerotiorum) on processing tomatoes. The experiment was performed in Goiânia, Brazil, with tomato hybrid Heinz 7155 in 2009 and 2010 in a field previously infested with S. sclerotiorum sclerotia. Treatments were arranged in a randomized complete block design in a 2 × 3 factorial structure (with and without Trichoderma spp. 1.0 × 10⁹ viable conidia mL⁻¹ ha⁻¹) × fluazinam (1.0 L ha⁻¹), procimidone (1.5 L ha⁻¹) and control, applied by drip irrigation. Treatments were applied three times 10 days apart, starting one month after transplanting. Each treatment consisted of plots with three 72-meter rows with four plants m⁻¹ and 1.5 m spacing between rows, with three replications. Based on disease incidence evaluated weekly, the area under the disease progress curve (AUDPC) was obtained. Yield and its components were evaluated in addition to fruit pH and °Brix. Results were subjected to ANOVA, Scott-Knott (5%), and regression analysis. Biocontrol using Trichoderma spp. via chemigation singly or in combination with synthetic fungicides fluazinam and procimidone reduced AUDPC and increased fruit yield up to 25 t ha⁻¹. The best treatment for controlling white mold also increased pulp yield around 1.0 and 7.0 t ha⁻¹ in 2009 and 2010, respectively. The present work demonstrated the advantages of white mold biological control in processing tomato crops, where drip irrigation favored Trichoderma spp. delivery close to the plants and to the inoculum source.     

Effects of drying methods on rheological properties of flaxseed gum

Flaxseed gum solutions were extracted and dried by different methods: ethanol precipitation, freeze drying, 105 °C oven drying, 80 °C oven drying, spray drying, and vacuum drying. The effects of these drying methods on the rheological properties of flaxseed gum were investigated in present study. Ethanol precipitation increased the apparent viscosity of flaxseed gum solution, while all the other methods decreased the apparent viscosity. Most of the drying methods slightly increased the activation energy, except ethanol precipitation. In frequency sweep test, all the drying methods reduced the G′ and G″ values. In creep–recovery tests, the data were modeled by Berger’s model. The E₂ and η₁ values were reduced by all of the drying methods in this study. Some relationships were found between the parameters in the Power Law model of the frequency sweep test and the parameters in Berger’s model.     

Effect of cryopreservation and microencapsulation of lactic acid bacterium Enterococcus faecium MC13 for long-term storage

The aim of this work was to investigate the effect of cryoprotectants on the survival of probiotic bacterium Enterococcus faecium MC13 during freeze drying and storage. The maximum relative cell viabilities were observed when cells were freeze dried and stored at −20 °C, which is optimum temperature for the preservation of E. faecium. At all storage temperatures, trehalose was found to be retaining the highest relative cell viability than other cryoprotectants. In addition, alginate–chitosan capsules were produced to encapsulate E. faecium with the aim of enhancing survival of probiotic cells and keeping the probiotic during exposure to the harsh gastro-intestinal conditions. Encapsulation of probiotic into alginate–chitosan capsules found to be retaining higher survival of probiotic cells (4.342 ± 0.26 Log CFU mL⁻¹) at −20 °C for six months. Microencapsulated cells were resistant to simulated gastric (pH 2.0) and intestinal fluids (pH 7.5), resulting in significantly enhanced survival when compared with free cells. During in vivo treatment, capsules were broken and probiotic cells were directly released into the intestinal tract of rat. This result showed that microencapsulation of E. faecium MC13 with alginate and a chitosan coating offers an effective means of delivery of viable cells to the colon and maintains their survival during the adverse gastro-intestinal conditions.     

Dry flowable formulations of antagonistic Bacillus subtilis strain T429 by spray drying to control rice blast disease

Dry flowable formulations of Bacillus subtilis strain T429 with fungicidal activity against the rice blast fungus Magnaporthe grisea were synthesized by spray drying. Inert ingredients including wetting agents, dispersants, disintegrants, and adhesives that show good biocompatibility with B. subtilis T429 were obtained. The formulations were optimized by a four-factor and three-level orthogonal experiment. The optimal contents of the wetting agent AEO-5, dispersant NNO, disintegrant (NH₄)₂SO₄, and adhesive CMC-Na were 1%, 9%, 5%, and 1% respectively, the filler kaolinite supplemented to 100%. The mixture was suspended in the fermentation broth of T429 at a ratio of 20% (m/v). After being ground in a ball mill for 3 h, the suspension was spray-dried, and the dry flowable formulations were obtained. The formulation showed good physical characteristics, such as high dispersibility and viability. After 12 months of storage at room temperature, it revealed long shelf life and high viability. Field tests in rice crops illustrated that dry flowable formulations at 50 and 75 g/667 m² concentrations were as effective as a commercial fungicide in controlling rice blast, control efficiency up to 77.6% and 78.5%, respectively. No significant differences in disease control efficiency were observed between the formulations and the chemical pesticide tricyclazole (79.5%). Overall, a new shelf-stable and effective dry flowable formulation of the biocontrol agent B. subtilis T429 was obtained by spray drying to control rice blast.     

The effect of the formulation on the shelf-life of biopesticides based on two colombian isolates of Trichoderma koningiopsis Th003 and Trichoderma asperellum Th034

BackgroundFour biopesticide prototypes formulated as dispersible granules and dry powders based on 2 Colombian isolates of Trichoderma koningiopsis (Th003) and T. asperellum (Th034) were developed. These microorganisms have antagonist activity against Fusarium oxysporum f. sp. lycopersici and Rhizoctonia solani with a reduction in incidence of between 70 and 100% in tomato crops and potato crops, respectively.AimTo determine the effect of the formulation on the shelf-life of 4 biopesticides based on T. koningiopsis Th003 and Trichoderma asperellum Th034 at 3 different temperatures.MethodsThe formulation effect was determined by evaluating the germination of unformulated and formulated conidia (dispersible granules and dry powder) stored at 8, 18 and 28 °C for 18 months. Germination kinetics were used to estimate the shelf-life by using different mathematical models (zero order, first order, second order, Higuchi model, Korsmeyer-Peppas model and polynomial model).ResultsThe products showed high stability of the conidia germination when they were stored at 8 and 18° C, with shelf-lives of 14.4 and 13.9 months for dry powder based on Th003, and 12.0 and 10.8 months for dry powder based on Th034, respectively. Prototypes formulated as dispersible granules stored at the same temperatures (8 and 18 °C) showed lower shelf-lives, with values of 11.6 and 10.9 months for the Th003 product, and 10.7 and 7.2 months for the dispersible granules based on Th034. Significant reductions in germination were observed on unformulated conidia at all storage temperatures evaluated.ConclusionsThe formulation type affected the conidia stability of the 2 Trichoderma spp. Colombian isolates. Dry powder was the prototype with the highest stability and shelf-life at all temperatures evaluated.     

Effects of wetting on the persistence of quiescent conidia of Isaria fumosorosea

Quiescent conidia of Isaria fumosorosea were submitted to various wetting–drying cycles under different regimes of temperature and air humidity. Germination and viability of conidia collected on cultures freshly host-passed (P2) were not affected at 25 °C during five cycles at increasing wet phase duration (2–12 h per daily cycle) under any moisture conditions (13–86% RH). Infectivity levels remained stable, but mortality was slightly postponed. In vitro-cultured inocula (P5) were significantly affected after only one cycle at higher air humidity (75 and 86% RH) and temperature (35 and 40 °C). The persistence of I. fumosorosea conidia suspended in water soluble extracts of leaf surfaces (corn and cabbage) confirmed the better persistence of P2 conidia and the relatively higher detrimental effect of lower air humidity conditions when combined with moderate temperatures. Quiescent conidia deposited in situ on potted plants of cabbage showed a higher persistence on wet foliage and on foliage submitted to wetting–drying cycles, than on dry foliage. These results underline that constraints prevailing in targeted environments and ecological fitness of fungal isolates have to be taken into account for assessing microbial control strategies.     

Evaluation of new fermentation and formulation strategies for a high endophytic establishment of Beauveria bassiana in oilseed rape plants

Since several years it is known that strains of the entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Ascomycota: Hypocreales) are able to colonize plants as a true endophyte. However, so far no integrated bioprocess engineering approaches have been published where fermentation and formulation strategies are combined to optimize colonization of oilseed rape plant tissues. We therefore aimed at investigating whether and how blastospore (BS) formation can be shifted to resilient submerged conidiospores (SCS) by introducing osmotic stress in different growth phases.When 50 g/L NaCl was added after 48 h to a culture of B. bassiana a yield of 1.4 ± 0.1 × 10¹⁰ SCS/g sucrose in shake flasks and 1.8 ± 0.1 × 10¹⁰ SCS/g sucrose in a stirred tank reactor were obtained. In a bioreactor, 24 h after the addition of NaCl, the formation of BS slowed down, the respiratory quotient decreased and a shift from BS to SCS set in.Following these steps, different formulation strategies, namely encapsulation, film coating and liquid formulation were evaluated. B. bassiana grew out of beads as well as on commercial fungicide-coated seeds. Due to the complete suppression of fungal growth on non-sterile soil, the most suitable option was a foliar application. A liquid formulation consisting of 0.1% Triton X-114, 1% molasses, 1% titanium dioxide and 10⁶ spores/mL was applied on leaf tips. After 14 days, the endophyte was detected by PCR and microscopic analysis in the leaves.Further research should focus on formation of SCS and protection of plants colonized by B. bassiana against herbivorous insects.     

Alginate embedding and subsequent sporulation of in vitro-produced Conidiobolus thromboides hyphae using a pressurised air-extrusion method

Conidiobolus thromboides is an entomophthoralean fungus with potential as a biological control agent of aphids. However, its application in biological control is limited due to its formulation requirements. The objective of this study was to develop and optimise a novel air-extrusion method to embed C. thromboides hyphae at high density in alginate pellets. An orthogonal experimental design was used to investigate selected combinations of parameters known to affect hyphal density within pellets. The diameter of pellets produced, and the calculated density of hyphae within them, ranged from 0.18 ± 0.09 to 3.17 ± 0.06 mm and from 0.02 to 350.56 mg/mm³ respectively. These data were used to predict the optimal parameter combination to deliver the greatest density of hyphae of C. thromboides per pellet: 1% sodium alginate, a 1:2 ratio of hyphae to sodium alginate, an orifice diameter of 0.232 mm and an air pressure of 0.05 MPa. Pellets made under the optimal conditions predicted produced a mean total of 4.3 ± 0.6 × 10⁵ conidia per pellet at 100% relative humidity which was significantly greater than the mean total number of conidia produced from infected aphid cadavers of comparable size (9.35 ± 0.85 × 10⁴) (p < 0.001). In conclusion, air-extrusion embedding appears to be a promising method for formulating in vitro-produced hyphae of C. thromboides for use in biological control.     

Nematicidal effect of volatiles produced by Trichoderma sp.

Trichoderma is an important biocontrol agent that produces metabolites harmful to nematodes. We investigated the volatile organic compounds (VOCs) of Trichoderma sp. YMF 1.00416 and examined their abilities to kill nematodes. Chemical investigations of the VOCs from this strain led to the isolation and identification of three metabolites: a new compound, 1β-vinylcyclopentane-1α,3α-diol (1) and two known metabolites, 6-pentyl-2H-pyran-2-one (2) and 4-(2-hydroxyethyl)phenol (3). Nematicidal activity assays showed that compound 2 was nematicidal, and killed > 85% of Panagrellus redivivus, Caenorhabditis elegans, and Bursaphelenchus xylophilus in 48 h at 200 mg/L in a 2 mL vial. Our results will help identify new nematicides.     

Strategies for controlling the ghost ant,Tapinoma melanocephalum (Hymenoptera: Formicidae) with liquid bait

The ghost ant, Tapinoma melanocephalum (F.), is a household pest and a considerable nuisance. The aim of this study is to demonstrate the toxicity and control efficacy of boric acid in liquid bait against queen and worker ghost ants. The LT₅₀ values for workers fed with 0.5%–2.5% boric acid and 2% chicken peptone in 20% sucrose water solutions were 4.3–2.4 days. The lethal times (LT₅₀ = 5.2–7.6 days) for queen ghost ants fed with various concentrations of a boric acid solution depended on the feeding behavior of the queens. The high boric acid (4%) content solutions were not repellent to the ghost ant workers. The liquid bait formulation of 1% boric acid, which caused a 100% worker, brood, and queen population reduction in 4, 4, and 5 weeks, respectively, was significantly more effective than the solid bait formulation containing the same concentration (p ≦ 0.05). The simulation tests involved using chicken peptone and sucrose as the attractant, and colonies were provided an alternate food source (20% sucrose solution and dog food) to achieve a more accurate assessment of bait acceptability in screening for the efficacy of the liquid boric acid bait. The control efficiency attained 99.9% in week 4. The results demonstrated that liquid bait, containing 2% chicken peptone, 20% sucrose as a food attractant, and 1% boric acid as the toxin, is efficient and highly recommended for ghost ant control.     

Effects of different inoculum densities of Trichoderma harzianum and Trichoderma viride against Meloidogyne javanica on tomato

A greenhouse experiment was conducted to evaluate the effects of different inoculum densities of two Saudi isolates of Trichoderma harzianum and Trichoderma viride against Meloidogyne javanica on tomato. Four densities (10⁴, 10⁶, 10⁸ and 10¹⁰ spores/g of soil) of each fungus were used. The results indicate that all four inoculum densities of the two Trichoderma species suppressed the nematode reproduction and root galling; and increased the growth of tomato plants, compared to controls. Efficacy of both fungi increased as their inoculum densities increased. Generally, efficacy of T. harzianum was better than that of T. viride, especially at the highest used density (10¹⁰ spore/g soil) which resulted in the best control.     

Functional characterization of the sucrose isomerase responsible for trehalulose production in plant-associated Pectobacterium species

Fifty-three plant-associated microorganisms were investigated for their ability to convert sucrose to its isomers. These microorganisms included one Dickeya zeae isolate and 7 Enterobacter, 3 Pantoea, and 43 Pectobacterium species. Eleven out of the 53 strains (21%) showed the ability to transform sucrose to isomaltulose and trehalulose. Among those, Pectobacterium carotovorum KKH 3-1 showed the highest bioconversion yield (97.4%) from sucrose to its isomers. In this strain, the addition of up to 14% sucrose in the medium enhanced sucrose isomerase (SIase) production. The SIase activity at 14% sucrose (47.6 U/mg dcw) was about 3.6-fold higher than that of the negative control (13.3 U/mg dcw at 0% sucrose). The gene encoding SIase, which is comprised a 1776 bp open reading frame (ORF) encoding 591 amino acids, was cloned from P. carotovorum KKH 3-1 and expressed in Escherichia coli. The recombinant SIase (PCSI) was shown to have optimum activity at pH 6.0 and 40 °C. The reaction temperature significantly affected the ratio of sucrose isomers produced by PCSI. The amount of trehalulose increased from 47.5% to 79.1% as temperature was lowered from 50 °C to 30 °C, implying that SIase activity can be controlled by reaction temperature.     

Succinic acid production from sucrose and molasses by metabolically engineered E. coli using a cell surface display system

To achieve sucrose-metabolizing capability, different sucrose utilization operons have been introduced into E. coli that cannot utilize sucrose. However, these engineered strains still suffer from low growth rates and low sucrose uptake rates. In this study, cell surface display system was adopted in engineered E. coli AFP111 for succinic acid production from sucrose and molasses directly. Invertase (CscA) from E. coli W was successfully anchored to outer membrane by fusion with OmpC anchoring motif, and the displayed CscA showed high extracellular activity. Compared with the sucrose permease system, the cell surface display system consumed less ATP during sucrose metabolism. When less ATP was consumed by AFP111/pTrcC-cscA, the succinic acid productivity from sucrose was 23% higher than that by AFP111/pCR2.1-cscBKA that having the sucrose permease system. As a result, 41 g L⁻¹ and 36.3 g L⁻¹ succinic acid were produced by AFP111/pTrcC-cscA from sucrose and sugarcane molasses respectively at 34 h in 3-L fermentor during dual-phase fermentation. In addition, 79 g L⁻¹ succinic acid was accumulated with recovered AFP111/pTrcC-cscA cells at the end of dual-phase fermentation in 3-L fermentor, and the overall yield was 1.19 mol mol⁻¹ hexose.     

Assessment of oral virulence against Spodoptera litura,acquired by a previously non-pathogenic Metarhizium anisopliae isolate,following integration of a midgut-specific insecticidal toxin

All entomopathogenic fungi infect insects by direct penetration through the cuticle rather than per os through the gut. Genetic transformation can confer fungi with per os virulence. However, unless the recipient isolate is nonpathogenic to the target insect, mortality caused by a transgenic isolate cannot be attributed solely to oral virulence due to the potential for some simultaneous cuticular infection. Here, a Metarhizium anisopliae wild-type isolate (MaWT) nonpathogenic to Spodoptera litura was genetically engineered to provide a transformed isolate (MaVipT31) expressing the insect midgut-specific toxin Vip3Aa1. Toxin expression was confirmed in MaVipT31 hyphae and conidia using Western blotting. Mortality, leaf consumption and body weight of S. litura larvae (instars I–IV) exposed to a range of concentrations of MaWT conidia were not significantly different to controls although the number of conidia ingested by surviving larvae during the bioassay ranged from 2.3 × 10⁵ (instar I) to 8.1 × 10⁶ (instar IV). In contrast, consumption of MaVipT31 conidia caused high mortalities, reduced leaf consumption rates and decreased body weights in all instars evaluated, demonstrating that oral virulence had been acquired by MaVipT31. Larval mortalities were much more dependent on the number of MaVipT31 conidia ingested than the duration of time spent feeding on conidia-treated leaves (r²: 0.83–0.94 for instars I–IV). LC₅₀ and LT₅₀ trends for MaVipT31 estimated by time-concentration-mortality modeling analyses differed greatly amongst the instars. For 50% kill to be achieved, instar I larvae required 3, 4 and 5 days feeding on the leaves bearing 103, 28 and 8 conidia/mm² respectively; instar IV larvae required 6, 7 and 8 days feeding on leaves bearing 1760, 730 and 410 conidia/mm² respectively. Our results provide a deeper insight into the high oral virulence acquired by an engineered isolate and highlight its great potential for biological control.     

Production of di-d-fructofranosyl-2,6′:2′,6-anhydride (DFA IV) by recombinant Bacillus subtilis carrying heterogenous levan fructotransferase from Arthrobacter nicotinovorans GS-9

We developed di-d-fructofranosyl-2,6′:2′,6-anhydride (DFA IV) production system with single culture of Bacillus subtilis directly from sucrose. This system can avoid the purification procedure of levan which organic solvent was used for precipitation. The levan fructotransferase (LFTase) gene was cloned from Arthrobacter nicotinovorans GS-9 (AHU1840, FERM P-15285) and expressed in levan producing B. subtilis 168. LFTase activity was detected in the culture supernatant of the transformant with maximal activity of 0.062 U/ml after 15.5 h post induction. Then sucrose was added as substrate and incubated. About 78 h after addition of sucrose, 20.5 g/l of DFA IV was produced from 139.3 g/l of sucrose consumed. The yield of DFA IV from sucrose was 14.7 wt.%.