In vitro regeneration from petiole explants of non-toxic Jatropha curcas

Jatropha curcas, a multipurpose shrub has acquired significant economic potential as biodiesel plant. The seeds or pressed cake is toxic due to the presence of toxic substances and is not useful as food/fodder despite having the best protein composition. A simple, efficient, and reproducible method for plant regeneration through direct organogenesis from petiole explants of non-toxic J. curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (57.61%), and number of shoot buds (4.98) per explant were obtained when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 μM TDZ. The Induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BA), and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation and subsequent elongation was achieved on MS medium supplemented with 2.25 μM BA and 8.5 μM IAA. The elongated shoots could be rooted on half-strength MS medium with 15 μM IBA, 11.4 μM IAA and 5.5 μM NAA with more than 90% survival rate.     

Tocochromanol content and composition in Jatropha curcas seeds

Physic nut (Jatropha curcas L.) is a promising seed oil source for biodiesel production. Natural antioxidants play a major role in maintaining oxidative stability of oils and they also have important food and industrial applications. Among them, tocochromanols are the most abundant in seeds. The objective of this research was to evaluate the variation for tocochromanol content and profile in a germplasm collection of 52 accessions of J. curcas. Seeds collected in two different periods, August and November of 2009, were analysed for tocochromanol content. Additionally, the dynamics of tocochromanol accumulation in developing seeds was studied. Total seed tocochromanol content averaged 307.2 mg kg⁻¹ in August and 303.7 mg kg⁻¹ in November, whereas total oil tocochromanol content averaged 507.4 mg kg⁻¹ in August and 500.8 mg kg⁻¹ in November. The tocochromanol fraction was made up of 15.4% gamma-tocopherol, 83.8% gamma-tocotrienol, and 0.8% delta-tocotrienol in August and 18.0% gamma-tocopherol, 80.4% gamma-tocotrienol, and 1.6% delta-tocotrienol in November. Genotype × environment effects were identified for tocochromanol content but not for the proportion of major tocochromanol homologues, which showed a high positive correlation between both environments. Developing seeds contained primarily alpha-tocopherol and gamma-tocopherol at early stages of development, with gamma-tocotrienol and delta-tocotrienol being practically undetectable. Gamma-tocotrienol content remained practically undetectable till 66 DAP and then increased pronouncedly to final levels of 177.1 mg kg⁻¹ (74.8% of the total tocochromanol content). The powerful antioxidant and health-promoting properties of gamma-tocotrienol encourages further studies on selection for the tocopherol/tocotrienol ratio in Jatropha and on the potential of tocochromanols as high added-value products derived from Jatropha seed oil production.     

Assessment of genetic stability in micropropagules of Jatropha curcas genotypes by RAPD and AFLP analysis

Jatropha curcas (Euphorbiaceae), a drought resistant non edible oil yielding plant, has acquired significant importance as an alternative renewable energy source. Low and inconsistent yields found in field plantations prompted for identification of high yielding clones and their large scale multiplication by vegetative propagation to obtain true to type plants. In the current investigation plantlets of J. curcas generated by axillary bud proliferation (micropropagation) using nodal segments obtained from selected high yielding genotypes were assessed for their genetic stability using Randomly Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) analyses. For RAPD analysis, 21 out of 52 arbitrary decamer primers screened gave clear reproducible bands. In the micropropagated plantlets obtained from the 2nd sub-culture, 4 out of a total of 177 bands scored were polymorphic, but in the 8th and 16th sub-cultures (culture cycle) no polymorphisms were detected. AFLP analysis revealed 0.63%, 0% and 0% polymorphism in the 2nd, 8th and 16th generations, respectively. When different genotypes, viz. IC 56557 16, IC 56557 34 and IC 56557 13, were assessed by AFLP, 0%, 0.31% and 0.47% polymorphisms were found, respectively, indicating a difference in genetic stability among the different genotypes. To the best of our knowledge this is the first report on assessment of genetic stability of micropropagated plantlets in J. curcas and suggests that axillary shoot proliferation can safely be used as an efficient micropropagation method for mass propagation of J. curcas.     

Predicting Jatropha curcas seed-oil content, oil composition and protein content using near-infrared spectroscopy—A quick and non-destructive method

The potential of near-infrared reflectance spectroscopy (NIRS) to estimate the oil content, fatty acid composition, and protein content of Jatropha curcas seeds was studied. Seventy-four intact kernels from various sources were scanned by NIRS. All samples were analyzed for oil content (hexane extractions), fatty acid composition (gas chromatography), and protein content (Kjeldahl). Calibration equations were developed for oil content, individual fatty acids (oleic C18:1, linoleic C18:2, stearic C18:0 and palmitic C16:0), and protein content. The performance of the calibration equations was evaluated through external and cross-validation. The results showed that NIRS was a reliable, accurate and nondestructive technique to estimate oil and protein contents, as well as oleic and linoleic fatty acid concentrations in J. curcas kernels; NIRS provides a rapid, simple, and cost-effective alternative method for screening intact J. curcas kernels.     

Improvements in the qualities of gluten-free bread after using a protease obtained from Aspergillus oryzae

From a nutritional perspective, rice flour is one of the most valuable flours and it is suitable for preparing food for people suffering from wheat allergy. However, bread made from rice flour is very difficult to bake because it lacks gluten. We found that pre-fermenting of rice flour using Aspergillus oryzae facilitated a better formulation of gluten-free rice bread. Bread swelling was remarkably improved with a longer pre-fermenting period at 55 °C. The specific loaf volume (SLV) without polymeric thickeners after a 12 h fermentation was approximately 2.2-fold (2.98 ml/g) higher than that after 0 h (1.36 ml/g). An enzymatic assay of the batter indicated that protease activity during the pre-fermentation period increased from 0.38 to 1.44 U/ml and this activity correlated with bread swelling. Furthermore, a commercial protease from A. oryzae also produced similar results with an adequate SLV of 3.03 ml/g. Rheological analysis showed that batter treated with protease had an increased batter viscosity and decreased flour settling behavior because of the aggregation of flour particle after partial cleavage of storage proteins. These results indicated that the improved SLV was mainly because of an A. oryzae protease, which affected the batter rheology thereby improving gas retention before baking.     

Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes

An efficient and reproducible protocol was established for genetic transformation in Jatropha curcas through microprojectile bombardment. Decotyledonated embryos from mature seeds were pre-cultured for 5 days and elongated embryonic axis was subjected to bombardment for the optimization of physical parameters. The frequency of transient gus expression and survival of putative transformants were taken into consideration for the assessment of physical parameters. Statistical analysis reveal that microcarrier size, helium pressure and target distance had significant influence on transformation efficiency. Among different variables evaluated, microcarrier size 1 μm, He pressure 1100 and 1350 psi with a target distance of 9 and 12 cm respectively were found optimum by co-relating microcarrier size, helium pressure and target distance on the frequency of gus expression and survival of putative transformants. Selection of putative transformants was done with increasing concentrations (5-7 mg L⁻¹) of hygromycin. The integration of desired gene into Jatropha genome was confirmed with PCR amplification of 0.96 and 1.28 kb bands of hptII and gus gene respectively from the T₀ transgenics and Southern blot analysis using PCR amplified DIG labeled hptII gene as a probe. A successful attempt of genetic transformation was made with optimized conditions using particle gene gun and establishing a stable transformation in J. curcas with 44.7% transformation efficiency. The procedure described will be very useful for the introgression of desired genes into J. curcas and the molecular analysis of gene function.     

Synthesis of Jatropha curcas oil-based biodiesel in a pulsed loop reactor

Jatropha curcas oil (JCO) has a high content of free fatty acids and has been used extensively as a feedstock in biodiesel production. In the present study, the transesterification reaction of JCO to Jatropha curcas methyl ester (biodiesel) was performed in a continuous pulsed loop reactor under atmospheric conditions. The JCO was pre-treated prior to the reaction to reduce the free fatty acid content to below 1% (w/w). The operating parameters of the loop reactor were optimised based on the conversion of the JCO to Jatropha curcas biodiesel and included reaction temperature, molar ratio of oil to MeOH, reaction time and oscillation frequency. The findings show that the highest reaction conversion of 99.7% (w/w) was achieved using KOH catalyst and 98.8% conversion was obtained using NaOCH₃ catalyst. The optimal operating conditions were a molar ratio of 6:1, an oscillation frequency of 6 Hz, temperature of 60 °C, feedstock FFA content of 0.5% (w/w) and only 10 min of reaction time. As a commercial commodity, the physical properties of biodiesel were analysed, and they compared well with the characteristics of fossil-based diesel fuel.     

Persistence and efficacy of spinosad on wheat,maize and barley grains against four major stored product pests

The insecticidal and residual effect of spinosad on wheat, maize and barley grain was evaluated in the laboratory against adults of Sitophilus oryzae (F.) (Coleoptera: Curculionidae), Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae), Tribolium confusum (DuVal) (Coleoptera: Tenebrionidae), Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae) as well as against larvae of T. confusum. Spinosad was applied as a solution to 2 kg lots of each commodity at three concentrations, 0.1, 0.5 and 1 ppm, and the treated grain quantities were kept at 25 °C and 65% RH. Samples were taken from each concentration-commodity combination at the day of storage and every 30 d for 6 consecutive months (6 bioassays). The test species were exposed for 14 d to the samples and mortality and reproduction were assessed over this exposure interval. With the exception of T. confusum, 1 ppm of spinosad was highly effective against the remainder of the tested species and provided protection for a period of storage at least 4 months. Although in general, spinosad performance was not very much affected by the grain type, efficacy on maize was less stable over the 6-month period of storage and declined sooner compared to the other commodities. Spinosad almost suppressed progeny production of R. dominica during the storage period, but did not suppress progeny of the other species, since progeny were recorded even 30 d post application especially with the lowest of the tested concentrations. The results of this study indicated that spinosad may provide suitable protection for 6 months against S. oryzae or R. dominica, but is not suitable for long-term protection against T. confusum or C. ferrugineus.     

Laboratory evaluation of diatomaceous earth deposits mined from several locations in central and southeastern Europe as potential protectants against coleopteran grain pests

Diatomaceous earth (DE) deposits from regions of central and southeastern Europe were evaluated for their insecticidal efficacy against Sitophilus oryzae (L.) (Coleoptera: Curculionidae), Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae) and Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) in comparison with the commercially available DE formulation SilicoSec. The effects of temperature, RH, grain commodity (wheat, barley, maize, rice), application method (spraying vs. dusting) were evaluated. FYROM, a DE from the Former Yugoslavian Republic of Macedonia, was the most effective of the DE deposits for grain treatment, whereas the least effective was from Greece (named Crete). However, for surface treatment, Slovenia was the most effective followed by Elassona 1 and Begora. Increase of temperature increased DE efficacy, while the reverse was noted with the increase of RH. Furthermore, the DEs were more effective in barley or wheat than in maize or rice. Neither the mined DEs nor SilicoSec were able to suppress progeny production of the tested species after previous exposure on the treated commodities. Generally, dust application of DEs was more efficacious than spraying against S. oryzae and T. confusum. However, spraying of wheat significantly reduced the bulk density (test weight) compared to dusting. For surface treatment, after 1 d of exposure, Slovenia was the most effective of the mined DEs followed by Elassona 1 and Begora, whereas after 6 d of exposure the mortality was almost complete (>99%) with all three DEs. More than 6 d of exposure were required for an effective control of T. confusum adults with the remainder of the mined DEs.     

Effects of fungicide seed treatments on germination, population, and yield of maize grown from seed infected with fungal pathogens

Seedborne fungi can reduce survival, growth, and yield of maize (Zea mays L.). Laboratory, field, and growth chamber experiments were conducted to determine the effects of the seed treatment fungicides fludioxonil, mefenoxam, and azoxystrobin on germination, plant population, and grain yield of maize grown from low-quality hybrid seed infected with seedborne fungal pathogens. Study I used seed of four hybrids infected at 0-54% incidence with Fusarium spp., Stenocarpella maydis, Penicillium spp., Rhizopus spp., and/or Aspergillus spp. Study II used three seed lots for each of two hybrids infected at 7-37% incidence with S. maydis. Warm and cold germination for untreated seed varied among hybrids in both studies. Warm germination of the seed lot with the highest incidence of S. maydis in study II treated with azoxystrobin and fludioxonil was significantly greater (+7%) than the nontreated control. Plant population in study I was significantly affected by seed treatment, hybrid, and their interactions. Populations were greater (≥9%) for fludioxonil, fludioxonil + mefenoxam, and fludioxonil + mefenoxam + azoxystrobin treatments compared to controls. In growth chamber experiments with pasteurized soil, emergence (≥5%) and plant dry weight (≥14%) were both greater than controls only with the triple seed treatment. Plant populations in study II for all seed treatments except mefenoxam and azoxystrobin alone were greater (≥4%) than controls. Yield in study I was significantly affected by hybrid and seed treatment. Yield for one hybrid was higher (≥20%) than the control with all seed treatments except fludioxonil, whereas yield with another hybrid was consistently greater (≥26%) only with the triple seed treatment. Yield in study II was significantly affected by hybrid, seed treatment, and their interactions. Yield was greater (≥8%) than the controls for all seed treatments with one hybrid and with all (≥5%) except azoxystrobin for the other hybrid. Highest yields occurred with the triple seed treatment. Results indicate that fludioxonil and azoxystrobin can increase germination, population, and yield of maize grown from seed infected by S. maydis and other fungi.     

Potato viruses in China

Potato (Solanum tuberosum L.) is globally the fourth largest staple crop after rice, wheat and maize. China is the largest potato producer worldwide, accounting for 26.3% and 22.2% of the global total area and yield, respectively. Viral diseases have been a limiting factor for sustainable production of potato in China. This article provides update and comprehensive information on the most important viruses, their effect on yield and their control in China. Meristem culture is currently the most widely used technique for production of virus-free potato plants and these plants are used as nuclear stock for the production of certified seed tubers. Although a significant increase in tuber yield has been achieved using certified seed potatoes, lack of application of a national seed potato production scheme has hindered progress. Conventional breeding using virus-resistant cultivars introduced from other countries as parents has already led to the production of Chinese cultivars. Use of virus-resistant wild Solanum species and genetic transformation techniques will provide new virus-resistant varieties in the future to further enhance control of potato viruses in China.     

Maritime Halophyte Species from Southern Portugal as Sources of Bioactive Molecules

Extracts of five halophytes from southern Portugal (Arthrocnemum macrostachyum, Mesembryanthemum edule, Juncus acutus, Plantago coronopus and Halimione portulacoides), were studied for antioxidant, anti-inflammatory and in vitro antitumor properties. The most active extracts towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were the methanol extracts of M. edule (IC₅₀ = 0.1 mg/mL) and J. acutus (IC₅₀ = 0.4 mg/mL), and the ether extracts of J. acutus (IC₅₀ = 0.2 mg/mL) and A. macrostachyum (IC₅₀ = 0.3 mg/mL). The highest radical scavenging activity (RSA) against the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical was obtained in the ether extract of J. acutus (IC₅₀ = 0.4 mg/mL) and H. portulacoides (IC₅₀ = 0.9 mg/mL). The maximum total phenolic content (TPC) was found in the methanol extract of M. edule (147 mg gallic acid equivalents (GAE)/g) and in the ether extract of J. acutus (94 mg GAE/g). Significant decreases in nitric oxide (NO) production were observed after incubation of macrophages with lipopolysaccharide (LPS) and the chloroform extract of H. portulacoides (IC₅₀ = 109 µg/mL) and the hexane extract of P. coronopus (IC₅₀ = 98.0 µg/mL). High in vitro cytotoxic activity and selectivity was obtained with the ether extract of J. acutus. Juncunol was identified as the active compound and for the first time was shown to display selective in vitro cytotoxicity towards various human cancer cells.     

Effect of plant growth promoting rhizobia on seed germination, growth promotion and suppression of Fusarium wilt of fenugreek (Trigonella foenum-graecum L.)

Five bacterial strains (TR1 to TR5) isolated from root nodules of fenugreek (Trigonella foenum-graecum) were tested for their plant growth promotory traits and biocontrol potential against Fusarium oxysporum. On the basis of morphological, physiological, biochemical and molecular characteristics, strains TR1 and TR3 - TR5 were identified as Ensifer meliloti, and TR2 as Rhizobium leguminosarum. All bacterial isolates utilized phosphate in vitro. Except TR5, all isolates produced IAA and none of them showed volatile cyanogens production. Except TR3, all isolates produced in vitro siderophore. Isolate TR1 and TR4 showed chitinase production while only TR2 showed β-1,3-glucanase activity. Isolates TR1, TR2 and TR5 exhibited ACC deaminase activity. Isolates TR1, TR2 and TR4 inhibited the growth of F. oxysporum, causing loss of structural integrity of the mycelium, hyphal perforation, lysis, fragmentation and degradation. The potential for nodulation and nitrogen fixation of the strains were confirmed by amplification of 500 bp nodC and 781 bp nifH fragments. The application of the TR1 + TR2 combination resulted in increased grain yield by 35% and 36% of fenugreek in two consecutive field trials, respectively as compared to control. Maximum increments in vigour index, nodule number and root and shoot biomass were recorded with seed inoculated with consortium (TR1 + TR2) followed by single inoculation as compared to control. The antibiotic resistant marker strain of E. meliloti TR1^strep+ and R. leguminosarum TR2^tet+ confirmed the efficient colonization of fenugreek roots. This study showed that these rhizobial isolates have properties of biocontrol agents and may be applied to promote the growth of fenugreek.     

Modeling the effects of solid state fermentation operating conditions on endoglucanase production using an instrumented bioreactor

Cellulase production is one of the most critical steps in the economics of second generation ethanol. Although solid-state fermentation (SSF) is an attractive process for the production of enzymes, SSF is highly limited by the difficulty in controlling the operating variables which affect microbial growth and metabolites production. In this context, this work evaluates the effects of operational conditions on endoglucanase production by a selected strain of Aspergillus niger cultivated under SSF using an instrumented lab-scale bioreactor equipped with an on-line automated monitoring and control system. The effects of air flow rate, inlet air relative humidity and substrate initial moisture on endoglucanase production were evaluated using a statistical design methodology. A correlation coefficient of 0.9106 and a calculated value of F, 5.46 folds higher than the listed value (P-value < 0.05) allowed the modeling of endoglucanase production under different process conditions. Higher endoglucanase production (56.1 U/g) was achieved for a selected condition of substrate initial moisture of 72%, air inlet humidity of 70%, and flow rate of 20 mL/min. A significant increase in endoglucanase production was also found to be achieved under forced aeration conditions (50.2 IU/g) compared to static conditions (29.8 IU/g) after 72 h of cultivation. Besides, respirometric analysis revealed that the total amount of CO₂ produced was linearly correlated with enzyme production (R² of 0.988). The bioreactor system used, as well as the methodology employed herein, was very effective in evaluating the influence of operational variables on enzymes production under SSF.     

Carbohydrate degrading enzyme production by plant pathogenic mycelia and microsclerotia isolates of Macrophomina phaseolina through koji fermentation

The ability of hydrolytic enzyme production by two different isolates of Macrophomina phaseolina was studied on apple pomace as a substrate for solid state fermentation (SSF). Initial moisture level, temperature and fermentation period was optimized so as to achieve higher output. Among the two different isolates, microsclerotial (MphP) and mycelial (MphM), MphP was observed as a potential source of different hydrolytic enzymes as compared to MphM. MphP gave higher enzyme activities (IU/gram dry substrate (gds): filter paper cellulase (FPase) activity 196.21 ± 16.3 (120 h), carboxymethyl cellulase (CMCase) 279.34 ± 28.25 (72 h), β-glucosidase (BGL) 129.82 ± 12.41 (96 h), xylanase 2527.88 ± 46.15 (120 h), and amylase 2780.72 ± 38.13 (96 h), respectively at 70% (v/w) IML. The incubation temperature was also found to have impact on the enzyme production ability of Macrophomina strains. The higher enzyme activities were achieved (IU/gds) as follows FPase 276.13 ± 25.02 (40 °C, 120 h), CMCase 278.11 ± 24.47 (35 °C, 144 h), BGL 189.47 ± 15.05 (30 °C, 144 h), xylanase 3845.77 ± 43.38 (35 °C, 144 h) and amylase activity of 3309.45 ± 29.22 (35 °C, 120 h), respectively using MphP at 70% (v/w) IML. This study reports for the first time the potential of carbohydrate degrading enzyme bioproduction by different isolates of M. phaseolina.     

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.     

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.     

Utilization of pretreated bagasse for the sustainable bioproduction of cellulase by Aspergillus nidulans MTCC344 using response surface methodology

Response surface methodology (RSM) was used to optimize the conditions for the production of endo β-1,4 glucanase, a component of cellulase by Aspergillus nidulans MTCC344 under solid state fermentation, using bagasse as the chief substrate. A four-factor-five-level central composite design was employed for experimental design and analysis of the results. Maximum cellulase activity (CMCase was 28.96 U g⁻¹) can be attained at the optimum conditions, 16.8 mm bagasse bed height, 60% moisture content, pH 4.25 and temperature 40 °C in the solid state fermenter. These data were rather close to the experimental results obtained (CMCase was 28.84 U g⁻¹). A. nidulans MTCC344 was able to hydrolyze pretreated bagasse completely after 8 days of incubation with significant endo β-1,4 glucanase activities. The results of Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) of bagasse showed structural changes through pretreatment, in favor of enzymatic hydrolysis. Bagasse with alkali pretreatment using sodium hydroxide is a source of lignocelluloses able to improve the yield of endo β-1,4 glucanase by the strain of A. nidulans. The endo β-1,4 glucanase produced during the bioconversion of cellulose to glucose by A. nidulans MTCC344 is strongly dependent on the pretreatment given before hydrolysis.     

Effects of crude seed extracts of Annona atemoya and Annona squamosa L. against the cabbage looper, Trichoplusia ni in the laboratory and greenhouse

Antifeedant, growth inhibitory and toxic effects of crude seed extracts of Annona squamosa and Annona atemoya from Fazenda Viveiro Bona, Parasisópolis – Minas Gerais, Brazil, were evaluated against the cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) using different bioassays. Crude methanolic seed extracts deterred feeding of third instar T. ni larvae in a leaf disc choice bioassay. A. squamosa was ∼10 times more active as a feeding deterrent than A. atemoya (DC₅₀ = 2.3 mg/ml vs. 20.1 mg/ml). A. squamosa was ∼three times more active as a growth inhibitor than A. atemoya (EC₅₀ = 38.0 ppm vs. 117.0 ppm). Methanolic seed extracts of A. squamosa and A. atemoya were toxic to third instar T. ni larvae both through topical and oral application. A. squamosa was more toxic through feeding (LC₅₀ = 167.5 ppm vs. 382.4 ppm) whereas, A. atemoya exerted greater toxicity via topical application (LC₅₀ = 301.3 μg/larva vs. 197.7 μg/larva). Both A. squamosa and A. atemoya extracts reduced leaf area consumption and larval growth in a greenhouse experiment. Our results indicate that both A. squamosa and A. atemoya have potential for development as botanical insecticides, especially for local use in Brazil.     

Study on antagonistic effects of Talaromyces flavus on Verticillium albo-atrum, the causal agent of potato wilt disease

Talaromyces flavus a fungal antagonist, was isolated from soil samples collected from potato fields in Varamin and Karaj districts, Tehran province, Iran. Antagonistic effects of T. flavus isolates against Verticillium albo-atrum, the causal agent of potato wilt disease were investigated in the laboratory and greenhouse conditions. T. flavus colonies were recovered after three weeks from soil samples cultured on selective medium. Antagonistic effects of volatile and non-volatile extracts of T. flavus isolates on V. albo-atrum growth were investigated in the laboratory and five that caused higher growth inhibition of V. albo-atrum, were selected for greenhouse experiments. Infection index was compared in the greenhouse in a split plot trial with five isolates applied to soil, seed, or both arranged in a randomized complete block design with four replications. The minimum infection index was observed when seed were treated with T. flavus with the most effective isolate being Tf-Po-V-52. On seed, the minimum infection index was observed with Tf-Po-V-50. The most effective T. flavus isolate was also evaluated in a field experiment. Results indicated that infection index in seed treatment contained this isolate was 0.15 whereas that of control was 3.5. The overall results of this study showed that it may be possible to manage potato Verticillium wilt disease effectively by using T. flavus, a biocontrol fungus.