Isolation of entomopathogenic Bacillus from a biodynamic olive farm and their pathogenicity to lepidopteran and coleopteran insect pests

The occurrence of Bacillus entomopathogenic bacteria on a Tunisian biodynamic farm was determined by examining 75 samples from olive tree (Olea europaea L.) habitats. A total of 40 Bacillus isolates were characterized according to their phenotypic, physiological and biochemical parameters. Isolates of the species Bacillus subtilis, Bacillus mycoides, Brevibacillus brevis, Paenibacillus polymyxa, Bacillus licheniformis, Bacillus sp. (1), Bacillus sp. (2) and a standard strain Btk HD-1 were used separately in feeding bioassays on fresh artificial diet against larvae of lepidopterans Prays oleae (Bernard) and Palpita unionalis (Hübner) and coleopterans Hylesinus oleiperda (F.) and Phloeotribus scarabaeoides (Bernard), which are olive tree pests. Larvae were successfully reared on an artificial diet with 25 g powdered olive tree leaves. Compared to the control data, only Btk and the isolates of B. licheniformis, P. polymyxa and B. brevis were entomopathogenic. Larval mortality assessed 7 days post-treatment showed high mortality rates with Btk to lepidopteran larvae (86.6% for P. oleae and 80.9% for P. unionalis) and low mortality against coleopteran pests. B. brevis isolates showed high mortality rates against P. oleae (up to 67.9%). B. licheniformis isolates caused up to 59.2% larval mortality for P. oleae and 43.6% for P. unionalis. Highest coleopteran mortality was achieved by P. polymyxa isolates (up to 55%). According to the 16S rDNA results, isolates of each of the three entomopathogenic strains were similar. Proteins in the strain supernatants were toxic to P. oleae larvae with LC50 values of 10.0 (B. brevis), 12.5 (B. licheniformis) and 37.6 μg/ml (P. polymyxa). Also, P. polymyxa showed an LC50 of 12.4 mg/l against P. scarabaeoides. Our results suggest that entomopathogenic Bacillus present locally in the biodynamic farm could be used in biological control programmes of olive tree pests.     

Biological control of Macrophomina phaseolina by chemotactic fluorescent Pseudomonas aeruginosa PN1 and its plant growth promotory activity in chir-pine

Ten strains of Pseudomonas aeruginosa (PN1 ˜ PN10) isolated from rhizosphere of chir-pine were tested for their plant growth promontory properties and antagonistic activities against Macrophomina phaseolina in vitro and in vivo. P. aeruginosa PN1 produced siderophore, IAA, cyanogen and solubilized phosphorus, besides producing chitinase and β-1,3-glucanase. In dual culture, P. aeruginosa PN1 caused 69% colony growth inhibition. However, cell free culture filtrate also posed inhibitory effect but to a lesser extent. After 90 days, P. aeruginosa PN1 increased plant growth and biomass in pots trial containing M. phaseolina-infested soil. PN1 showed the strong chemotaxis toward root exudates resulting in effective root colonization. Moreover, increased population in rhizosphere of these bacteria was also recorded after 90 days of treatment. Thus, chemotactic fluorescent P. aeruginosa PN1 exhibited strong antagonistic property against M. phaseolina, suppressed the disease and improved plant growth of the seedlings of chir-pine proving potential biocontrol agent.     

Actinomycetes mediated biochemical responses in tomato (Solanum lycopersicum) enhances bioprotection against Rhizoctonia solani

Six actinomycetes isolates, namely Streptomyces toxytricini vh6, Streptomyces flavotricini vh8, S. toxytricini vh22, Streptomyces avidinii vh32, Streptomyces tricolor vh85 and vh41, an isolate of an unknown species of Actinomycetales, were tested for their efficacy in protecting tomato (Solanum lycopersicum) against Rhizoctonia solani under green house conditions. Actinomycetes treated plants showed better growth in terms of high chlorophyll content, higher phenylalanine ammonia lyase (PAL) activity and high total phenolic content. Qualitative and quantitative estimation of phenolic compounds from tomato leaves showed significant accumulation of six phenolic acids, gallic (29.02 μg g⁻¹ fresh leaf wt), ferulic (11.44 μg g⁻¹ fresh wt), cinnamic (56.84 μg g⁻¹ fresh wt), gentisic (24.19 μg g⁻¹ fresh wt), chlorogenic acid (1.72 μg g⁻¹ fresh wt) and salicylic (0.39 μg g⁻¹ fresh wt) acid, in actinomycetes treated plants. Biochemical profiling, when correlated with plant mortality in actinomycetes treated and untreated plants, indicated that isolates vh6 and vh8 offered 44.55% and 40.14% disease reductions, respectively compared to the control. These results established that these organisms have the potential to act as biocontrol agents.     

Management of the root-knot nematode Meloidogyne incognita on tomato with combinations of different biocontrol organisms

The nematicidal effect of Pseudomonas fluorescens, Paecilomyces lilacinus, Pichia guilliermondii and Calothrix parietina singly or in combination was tested against root-knot nematode, Meloidogyne incognita. Treatments with P. fluorescens and P. lilacinus caused mortality of M. incognita as 45% and 30% of juveniles after 48 h of exposures, respectively compared to water control in vitro. Under greenhouse conditions, all treatments reduced the disease severity and enhanced plant growth compared to untreated control. Application of P. fluorescens, P. lilacinus and P. guilliermondii Moh 10 was more effective compared to C. parietina. There was a negative interaction between C. parietina and either P. lilacinus or P. guilliermondii. Fresh and dry weight of shoots and roots of plants were significantly reduced as a result of infection with M. incognita, however application of biocontrol agents singly or in mix recovered this reduction. Moreover, they enhanced the growth parameters compared with the control. Our results proved that application of different biocontrol agents (P. fluorescens, P. lilacinus and P. guilliermondii) not only has a lethal effect on nematode, but also enhances the plant growth, supplying many nutritional elements and induction the systemic resistance in plants. Presence of C. parietina as a soil inhabitant cyanobacterium could antagonize biocontrol agents leading to the reduction of their practical efficiency in soil.     

Wilt disease management and enhancement of growth and yield of Cajanus cajan (L) var. Manak by bacterial combinations amended with chemical fertilizer

Root nodulating Sinorhizobium fredii KCC5 and Pseudomonas fluorescens LPK2 were isolated from nodules of Cajanus cajan and disease suppressive soil of tomato rhizosphere, respectively. Both strains produced IAA, siderophore, solubilized insoluble phosphate, showed chitinase and β-1,3-glucanase activities, and strongly inhibited the growth of Fusarium udum. It also caused degradation and digestion of cell wall components, resulting in hyphal perforations, empty cell (halo) formation, shrinking and lysis of fungal mycelia along with significant degeneration of conidia. LPK2 produced volatile cyanogen (HCN). Combinations of S. fredii KCC5 and P. fluorescens LPK2 with half dose of chemical fertilizers showed a significant increase in seed germination (94%) while seed germination with co-inoculated strains (KCC5 + LPK2), KCC5 and LPK2 alone was 90, 84 and 82% respectively as compared to control 77%. After 120 days of sowing, per plant number of pods, nodules, shoot length, root length, shoot weight and root weight were greater for the combination with half dose of chemical fertilizers compared to the control. Combinations of S. fredii KCC5 and P. fluorescens LPK2 with half dose of chemical fertilizers resulted in an 82% increase in grain yield per hectare compared to the control. Both strains KCC5 and LPK2 led to proto-cooperation as evidenced by synergism, aggressive colonization of the roots, and enhanced growth, suggesting potential biocontrol efficacy against Fusarium wilt in C. cajan.     

Sensitivity of Ascochyta rabiei populations to prothioconazole and thiabendazole

Ascochyta rabiei causes Ascochyta blight, a yield-limiting disease of chickpea (Cicer arietinum) world-wide. In 2007, fungal populations of A. rabiei resistant to the QoI group of fungicides were detected in the Northern Great Plains of the United States. Assays were conducted to determine fungal sensitivity for two alternative fungicidal modes of action. A total of 78 isolates of A. rabiei collected between 1983 and 2007 were screened to determine baseline sensitivity to the demethylation-inhibiting foliar fungicide, prothioconazole, and 100 isolates collected between 1987 and 2007 were screened for sensitivity to the methyl benzimidazole carbamate (MBC) fungicide, thiabendazole. Isolates were tested using an in vitro mycelial growth assay to determine the effective fungicide concentration at which 50% of fungal growth was inhibited (EC₅₀) for each isolate-fungicide combination. Baseline EC₅₀ values of prothioconazole ranged from 0.0526 to 0.2958 μg/ml, with a mean of 0.1783 μg/ml. Isolates of A. rabiei collected from 2007 to 2009 from North Dakota chickpea fields exposed to prothioconazole, were screened for prothioconazole sensitivity using the same assay. Mean EC₅₀ values for these isolates were 0.3544 μg/ml, 0.3746 μg/ml, and 0.7820 μg/ml, respectively. These values represent an approximate 2.0 (2007-2008) and 4.4-fold (2009) decrease in sensitivity from the baseline mean. EC₅₀ values of thiabendazole ranged from 1.192 to 3.819 μg/ml, with a mean of 2.459 μg/ml. No significant decrease in fungicide sensitivity was observed for thiabendazole. To date, no loss of Ascochyta blight control has been observed with the use of either prothioconazole or thiabendazole.     

Mycoparasitic Trichoderma viride as a biocontrol agent against Fusarium oxysporum f. sp. adzuki and Pythium arrhenomanes and as a growth promoter of soybean

Trichoderma viride was proved as an effective biocontrol agent against two fungal pathogens, Fusarium oxysporum f. sp. adzuki and Pythium arrhenomanes, infecting soybean. During an in vitro biocontrol test, Trichoderma showed mycoparasitism and destructive control against the tested fungal pathogens. Both the pathogens significantly influence the germination and P. arrhenomanes had a severe effect (only 5% germination). The root system of the soybean plant was poorly developed due to the infection and it exerted a negative influence on the nodulation and further growth phases of the plant. During pot assay along with biocontrol activity, Trichoderma showed growth promoting action on the soybean plant. Trichoderma enhanced growth of shoot and root systems and fruit yield after 12 weeks of growth. Pythium and Fusarium infected plants treated with Trichoderma had ∼194% and 141% more height than pathogens alone. The fruit yield treated with Trichoderma was ∼66 per plant whereas the yield was only 41 for a control plant. The plants infected with Pythium and Fusarium and treated with Trichoderma had fruit yields of 43 and 53 respectively and those were 5 and 1.6 times higher than plants infected with pathogens.     

Biocontrol of Rhizoctonia solani and Sclerotium rolfsii on tomato by delivering antagonistic bacteria through a drip irrigation system

In a 2-year assessment carried out on tomato crops of Central and Southern Italy, a high incidence of Rhizoctonia solani and Sclerotium rolfsii was found. These fungal pathogens attack horticultural crops and are responsible for severe crown and stem rot. Because of technical, economical and environmental issues their chemical control is an arduous task. To find alternative and eco-compatible control methods, the effectiveness of two new antagonistic bacterial isolates (Burkholderia cepacia, T1A-2B, and Pseudomonas sp., T4B-2A), previously selected from suppressive organic amendments, were tested on tomato plants grown under both growth chamber and field conditions. The potential antagonists were compared with two commercial biofungicides, based on Bacillus subtilis (BSF4) and Trichoderma asperellum (TV1), and four synthetic fungicides (tolclofos-methyl, azoxystrobin, fosetyl-Al and fosetyl-Al + propamocarb). In 2-year field experiments carried out on tomato plants, the biocontrol bacteria as well as the other treatments were applied to the soil, proximal to the plant crowns and main roots, by means of an effective and specific system of drip irrigation. In all the experiments the novel selected biocontrol bacteria significantly reduced both incidence and severity of the diseases caused by S. rolfsii or R. solani, with results demonstrating effectiveness equal to TV1, better than BSF4 and comparable with the synthetic fungicides, except for tolclofos-methyl which was the most effective treatment. In field experiments, carried out for two consecutive years, isolate T1A-2B reduced up to 58.33% and up to 63.8% the severity of the diseases caused by S. rolfsii and R. solani respectively; whereas isolate T4B-2A gave reduction of S. rolfsii and R. solani diseases severity up to 73.2% and up to 62.7%, respectively.     

Fungicidal activity of fluopicolide for suppression of Phytophthora capsici on squash

The efficacy of a new fungicide fluopicolide in suppression of Phytophthora blight caused by Phytophthora capsici was evaluated under laboratory and field conditions. Studies with 51 P. capsici isolates from vegetable crops in Georgia, USA, indicated that 5.9% of the isolates were resistant, 19.6% were intermediately sensitive, and 74.5% were sensitive to 100 μg ml⁻¹ of mefenoxam based on in vitro mycelial growth. EC₅₀ values of fluopicolide in inhibiting mycelial growth of 25 isolates, representing resistant, intermediately sensitive, and sensitive to mefenoxam, ranged from 0.05 to 0.35 μg ml⁻¹ with an average of 0.2 μg ml⁻¹ EC₅₀ values of fluopicolide in suppressing zoospore germination and sporangium production of the 25 isolates ranged from 1.1 to 4.5 μg ml⁻¹ and 0.3–9.0 μg ml⁻¹, respectively. Evaluation of a collection of 150 P. capsici isolates from vegetables and irrigation ponds found none of the isolates were resistant to 10 μg ml⁻¹ of fluopicolide. Field experiments were conducted to determine the efficacy and application methods of fluopicolide for control of P. capsici on squash in spring 2007 and 2009. Fluopicolide applied through drip irrigation or as a foliar spray at 86.6 or 115.4 g ha⁻¹ consistently provided significant disease reduction and increased squash yield. Results with fluopicolide were similar or slightly superior to mefenoxam applied at recommended rate. Fluopicolide applied at 57.7 g ha⁻¹ did not provide consistent satisfactory disease suppression. The results indicated that fluopicolide was effective in suppression of different stages of the life cycle of P. capsici and could be a viable alternative to mefenoxam for managing Phytophthora blight in squash production.     

Two Bacillus amyloliquefaciens strains isolated using the competitive tomato root enrichment method and their effects on suppressing Ralstonia solanacearum and promoting tomato plant growth

Two tomato root colonizing strains, Bacillus amyloliquefacien CM-2 and T-5, were isolated after the enrichment procedure on the roots of tomato seedlings, and evaluated for their antagonistic activities against pathogenic Ralstonia solanacearum (RS) in vitro. Three inoculation methods were used to test their biocontrol efficacy and growth promotion effects in greenhouse based on their colonization ability in rhizosphere soil. Both CM-2 and T-5 strains showed strong biocontrol and growth promotion effects on tomato seedlings. The best biocontrol efficacy was obtained by treating both seedlings and soils with the biocontrol agents. In comparison to the control, the disease incidence was reduced by 70.1 and 79.4% for CM-2 and T-5, respectively. The numbers of colony-forming units of RS in rhizosphere soil were significantly (P < 0.05) decreased as compared to the control. The density of both strains in the rhizosphere soils remained at a high level (≥10⁷ CFU/g) during a five-week period. Both strains were recovered from the interior of the stems and roots of plants. They showed positive reactions for ammoina, indole acetic acid and siderophores production, and phosphate solubilizing activity. Our data proved the potential of isolated strains for the biocontrol of tomato bacterial wilt.     

Occurrence and characterization of fungi and oomycetes transmitted via potting mixtures and organic manures

A study was conducted to investigate the most common fungal and oomycete pathogens introduced into farms in Oman via potting mixtures and organic manures. A total of 37 commercial types of potting mixtures (2 local and 35 imported from overseas), 4 commercial types of organic manures and 11 non-commercial types of organic manures were included in the study. Identification of the isolated species was based on morphological characteristics, except for the most common species which were further identified using sequences of the internal transcribed spacer region of the ribosomal DNA (ITS rDNA). Fusarium spp. (14%), Pythium aphanidermatum (3%), Alternaria spp. (5%), Helminthosporium spp. (5%) and Cladosporium spp. (3%) were recovered at different frequencies from samples of potting mixtures. Fusarium solani (40%) and Fusarium equiseti (47%) were recovered at high frequencies from samples of organic manures. Isolations from organic manures also yielded Pythium periplocum (7%), Rhizoctonia solani (7%), Fusarium lichenicola (7%), Helminthosporium spp. (27%) and Alternaria spp. (27%). Trichoderma spp., Penicillium spp., Aspergillus spp. and Rhizopus spp. were found to be common in samples of potting mixtures and organic manures. Investigating sensitivity to hymexazol among 9 isolates of F. equiseti and 13 isolates of F. solani revealed variations among different isolates. The EC₅₀ values ranged from 1 to over 1200 (avg. 192 μg ml⁻¹) for F. equiseti isolates and from 135 to 789 (avg. 324 μg ml⁻¹) for F. solani isolates, indicating presence of resistance to this important fungicide among some Fusarium isolates. This appears to be the first report of contamination with R. solani, P. periplocum, F. solani, F. equiseti and F. lichenicola of organic manures. This study appears to report for the first time F. lichenicola in Oman and appears to be the first report of occurrence of resistance to hymexazol among F. equiseti and F. solani isolates.     

Effects of middle-viscosity chitosan on Ramularia cercosporelloides

The inhibitory properties of middle-viscosity Chitosan on the growth of fungus Ramularia cercosporelloides were studied in vitro. The inhibitory concentration that delayed 50% of the radial growth (IC₅₀) was 3.4 g l⁻¹ for middle-viscosity Chitosans with molecular weights of about 133 and 187 kDa (middle molecular weight) dissolved in lactic and acetic acids, respectively. At 96 h of incubation and under the same growing conditions, inhibitions of 91.79% and 73.13% of the radial growth of the fungus were observed when 3.4 g l⁻¹ of Chitosan was dissolved in 0.05 M acetic and 0.05 M lactic acid, respectively. The biomass production was significantly lower than that observed in the controls at 72 h. Based on these in vitro results; Chitosan could be a good alternative to control the disease caused by R. cercosporelloides on safflower.     

Population dynamics of Xanthomonas campestris pv. vitians on different plant species and management of bacterial leaf spot of lettuce under greenhouse conditions

The population dynamics of Xanthomonas campestris pv. vitians (Xcv) was studied both externally and internally in lettuce, tomato and pepper plants. In addition, the application of bactericides during transplant production period was carried out for the management of bacterial leaf spot of lettuce under greenhouse conditions. Epiphytic populations of Xcv were recovered on leaves of lettuce plants (10⁵ CFU/g) 5 weeks after sprayed than the other plant species when inoculated with 10⁸ CFU/ml of Xcv. When plants of each crop species infiltrated with the bacterium at 10⁵ CFU/ml, the highest populations were developed in lettuce (10⁸ CFU/cm²) followed by pepper with 10⁶ CFU/cm² and tomato with 10⁵ CFU/cm² 10-days after infiltration. Application of a mixture of Maneb and Kocide or Kocide alone as a foliar spray on lettuce significantly reduced the incidence and disease severity of bacterial leaf spot by 29 and 27% respectively. Spread of the bacterium and development of the disease may partly be managed by avoiding intercropping of these plants commonly grown in close proximity to lettuce. For the management of leaf-associated populations of Xcv in lettuce, use of a mixture of Maneb and Kocide is advocated to minimize the effect of attacks.     

Evaluation of actinomycete isolates obtained from herbal vermicompost for the biological control of Fusarium wilt of chickpea

A total of 137 actinomycetes cultures, isolated from 25 different herbal vermicomposts, were characterized for their antagonistic potential against Fusarium oxysporum f. sp. ciceri (FOC) by dual-culture assay. Of the isolates, five most promising FOC antagonistic isolates (CAI-24, CAI-121, CAI-127, KAI-32 and KAI-90) were characterized for the production of siderophore, cellulase, protease, hydrocyanic acid (HCN), indole acetic acid (IAA) and antagonistic potential against Rhizoctonia bataticola, which causes dry root rot in chickpea (three strains viz. RB-6, RB-24 and RB-115) and sorghum (one strain). All of the five FOC antagonistic isolates produced siderophore and HCN, four of them (except KAI-90) produced IAA, KAI-32 and KAI-90 produced cellulase and CAI-24 and CAI-127 produced protease. In the dual-culture assay, three of the isolates, CAI-24, KAI-32 and KAI-90, also inhibited all three strains of R. bataticola in chickpea, while two of them (KAI-32 and KAI-90) inhibited the tested strain in sorghum. When the FOC antagonistic isolates were evaluated further for their antagonistic potential in the greenhouse and wilt-sick field conditions on chickpea, 45-76% and 4-19% reduction of disease incidence were observed, respectively compared to the control. The sequences of 16S rDNA gene of the isolates CAI-24, CAI-121, CAI-127, KAI-32 and KAI-90 were matched with Streptomyces tsusimaensis, Streptomyces caviscabies, Streptomyces setonii, Streptomyces africanus and an identified species of Streptomyces, respectively using the BLAST searching. This study indicated that the selected actinomycete isolates have the potential for biological control of Fusarium wilt disease in chickpea.     

The effects of nitrogen rate and the ratio of NO3:NH4 on Bemisia tabaci populations in hydroponic tomato crops

Nitrogen fertilization is one of the factors that influences Bemisia tabaci (Gennadius) population density. The aim of this study was to determine the effects of three N application rates (75, 205 and 335 mg/l) and three ratios of NO₃⁻:NH₄⁺ ions (92:8, 75:25 and 55:45) in standard nutrient solution (205 mg/l N) on the population density of B. tabaci. The experiments were conducted on spring-summer hydroponic crops of tomato. The effect of plant stratum on the whitefly population was also determined. The aggregation of B. tabaci adults as well as their oviposition rate was higher at 205 and 335 mg/l N than on plants grown at 75 mg/l N. By the end of the experiment (60 d after infestation), the number of nymphs on plants at 205 mg/l N was higher than on plants at 75 mg/l N. The number of pupae was lowest on plants supplied with 75 mg/l N. An increase in NH₄⁺ percentage in standard nutrient solution (from 25% to 45% of the total N) reduced adult population density and oviposition rate. The density of nymphs and pupae, at 60 d after infestation, was lower on the tomato plants grown at 75:25 and 55:45 NO₃⁻:NH₄⁺ ratios compared to the 92:8 ratio. The 75:25 and 55:45 NO₃⁻:NH₄⁺ ratios resulted in a higher incidence of blossom-end rot of tomato fruit, with a lower incidence of disorder at 75:25 than at the 55:45 ratio. Plant stratum influenced adult whitefly distribution in two years of the study. Middle stratum leaves were more attractive to adults in both years. The results demonstrate the effects of N fertilization (N rate and the ratio of NO₃⁻:NH₄⁺) and plant stratum on B. tabaci population density.     

Insecticidal activities of crude extracts and phospholipids from Chenopodium ficifolium against melon and cotton aphid, Aphis gossypii

Several organic solvent extracts of Chenopodium ficifolium were tested for their insecticidal activity against melon and cotton aphid, Aphis gossypii, on cucumber plants. Both methanol and ethanol extracts, at 5000 μg ml⁻¹, were highly active giving over 80% control. The other crude extracts displayed moderate or weak insecticidal activity giving control in the range of 16–69%. Two phospholipids were isolated as insecticidal active substances from C. ficifolium. Their chemical structures were identified as 1-palmitoyl-2-(3-trans)-hexadecenoyl-sn-glycero-3-phosphoglycerol and 1-palmitoyl-2-linoleoyl-3-glycerophosphocholine by GC–MS, EDS, mass and NMR spectral analyses. Both compounds displayed a dose-dependent mortality of A. gossypii. Furthermore, the liquid formulation that was obtained by partitioning with n-hexane from the methanol extract of C. ficifolium controlled melon and cotton aphid on cucumber plants effectively. These results indicate that extracts of C. ficifolium have potential for development as botanical insecticides for controlling A. gossypii infesting cucumber plants.     

Effect of the use of dilute alkaline prior to Bacillus subtilis-based biocontrol steeping and germination conditions on red sorghum malt β-glucanase activities and residual β-glucans

Malting is the ideal stage to deal with β-glucans. Their hydrolysis is very important as the diffusion of both hormones and hydrolytic enzymes in the endosperm of germinated grain depend on it. A high malt β-glucanase activity is not a guarantee of an extensive hydrolysis of β-glucans. When Bacillus subtilis is used to control mould growth, red sorghum malt β-glucanase activity (measured using carboxymethylcellulose as the substrate) was improved without significantly affecting the hydrolysis of malt β-glucans. Thus, in order to reduce the residual β-glucans content, soaking in 0.2% NaOH was combined with a biocontrol. Soaking in 0.2% NaOH is recognized as capable of improving grain hydration by opening-up the endosperm cell walls. The combined use of 0.2% NaOH with B. subtilis-based biocontrol treatments during red sorghum malting, leads to malt with increased β-glucanase activity and a significant reduction of residual β-glucans when compared with the 16 h biocontrol steeping without prior steeping in 0.2% NaOH. β-glucanase activity increases with increased germination temperature and time while, conversely, the residual β-glucans content of the malts decreases. Indeed, while the level of β-glucanase was not vastly different between the malts obtained after steeping in distilled water and those obtained after 8 h steeping in 0.2% NaOH followed by 8 h resteeping in distilled water (NaOH + H₂O treatment), their residual β-glucans levels differ significantly. B. subtilis-based treatment leads to malt with improved β-(1-3)- and β-(1-4)-glucanase activities without significantly improved malt β-(1-3),(1-4)-glucanase activity. While malts obtained after 84 h germination weren't significantly different in terms of malt β-(1-3),(1-4)-glucanase activities for all steeping treatments, the use of 0.2% NaOH steeping prior to resteeping led to malts with improved β-glucans content. Combining the steeping in dilute alkaline and biocontrol enables taking advantage of the dilute alkaline effect on residual β-glucans content, due probably to the opening-up of the cell walls and the improvement of water uptake, and that of the biocontrol (improvement of β-glucanase synthesis).     

Time-mortality relationships of larvae and adults of grain beetles exposed to extreme cold

Mortality effects of low temperature exposure to −16 °C were investigated on larvae and adults of the grain beetles Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae), Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) and Trogoderma granarium (Everts) (Coleoptera: Dermestidae), in the laboratory. The main effects and interactions of exposure time (0.2, 0.5, 1, 2, 4, 8, 12, 24 and 48 h), developmental stage (larva and adult) and individual’s age (young and old) were examined. After 4 h of exposure 100% mortality has been achieved in most of the treatments. The only exceptions were that of old larvae of T. granarium and O. surinamensis, and adults of T. granarium which needed 48, 8, and 24 h, respectively. T. granarium was the most cold-hardy species irrespective of developmental stage, and age, followed in decreasing order by O. surinamensis and T. confusum. Younger adults were generally more susceptible to cold in the cases of O. surinamensis and T. confusum. On the contrary, older adults of T. granarium suffered higher mortality than younger ones. Larvae of T. granarium were generally more cold-tolerant than adults but the opposite pattern was observed in O. surinamensis and T. confusum. Main effects of exposure time, developmental stage and individual’s age on mortality proved to be significant for all species, with the exception of T. granarium where the effect of developmental stage proved to be insignificant. Lethal time values were estimated via probit analysis. Values of LT₅₀ ranged between 0.7 and 1.0 h for T. confusum, 0.7-1.9 h for O. surinamensis and 1.2-3.4 h for T. granarium. Our results are discussed with findings from relevant studies and the possibility of including extreme cooling to IPM programs against stored product pests is investigated.     

Comparative susceptibility of Ostrinia furnacalis, Ostrinia nubilalis and Diatraea saccharalis (Lepidoptera: Crambidae) to Bacillus thuringiensis Cry1 toxins

Transgenic corn hybrids that express toxins from Bacillus thuringiensis (Bt) are highly effective against the European corn borer, Ostrinia nubilalis (Hübner), and the closely related Asian corn borer, Ostrinia furnacalis (Guenée). Since the registration of Bt corn hybrids in the U.S. in 1996, there has been a great deal of information generated on O. nubilalis. However, relatively little information exists for O. furnacalis. To help determine whether the information generated for O. nubilalis can be leveraged for decisions regarding the use of transgenic Bt corn against O. furnacalis, experiments were designed to determine whether the pattern of sensitivity to various Bt Cry1 toxins is similar between the two species. Test insects included laboratory-reared O. furnacalis originating from Malaysia, a Bt-susceptible laboratory colony of O. nubilalis maintained at the University of Nebraska-Lincoln (UNL) and an out-group consisting of the sugarcane borer, Diatraea saccharalis (F.), from Louisiana which represents a different genus from the same family. O. furnacalis and O. nubilalis exhibited a similar pattern of susceptibility to all the Cry1 toxins and were highly susceptible to the range of Bt toxins tested including Cry1Aa, Cry1Ab, Cry1Ac and Cry1F. Both of the Ostrinia species were more tolerant to Cry1Ba compared with D. saccharalis, although sensitivity of O. furnacalis was intermediate and did not differ significantly from that of O. nubilalis and D. saccharalis. D. saccharalis was also susceptible to the range of toxins tested but unlike the two Ostrinia species, was more tolerant to Cry1F and more susceptible to Cry1Ba. These results indicate that both of the Ostrinia corn borer species are similar in sensitivity to the Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1F toxins, thus suggesting shared toxin receptors and mechanisms of toxicity for the two species.     

Evaluations of shallot genotypes for resistance against fusarium basal rot (Fusarium oxysporum f. sp. cepae) disease

Fusarium basal rot (FBR) caused by Fusarium oxysporum f. sp. cepae (Foc) is one of the most significant production constraint to shallot. Field experiment was conducted in a naturally Foc infested soil at Debre Zeit Agricultural Research Center during 2006 and 2007 cropping seasons to evaluate the level of resistance of sixteen shallot genotypes against FBR disease. Treatments were arranged in randomized complete block design with four replications. The genotypes significantly varied in their susceptibility to FBR and yield. They were grouped into tolerant, moderately and highly susceptible types. Five genotypes (DZ-Sht-168-1A, DZ-Sht-157-1B, Huruta, Negelle and DZ-Sht-169-1b) were identified to be tolerant as they had reduced disease severity levels from 26.8 to 32.5% and increased mean yield by more than 5 t ha⁻¹ compared to highly susceptible genotypes (DZ-Sht-076-4, DZ-Sht-201-1C and DZ-Sht-054-3A). Among the tolerant genotypes, DZ-Sht-169-1b had greatly reduced bulb rot incidence by 48% in ground storage and 30% in wire mesh shelf as compared to highly susceptible genotype DZ-Sht-201-1C. The tolerant genotypes have high yielding characteristic, and farmers could adopt them for cultivation where FBR is a problem.