Bacterial Antagonist as Seed Treatment to Control Leaf Blight Disease of Bambara Groundnut (Vigna subterranea)

Summay Soil samples were taken from 48 fields in the southern part of Thailand in which either bambara groundnut (Vigna subterranea) or groundnut (Arachis hypogeae) had been planted. Bacillus spp. were isolated using soil dilution plates and heat treatment to screen for endospore-producing bacteria. Among 342 Bacillus spp. isolates tested, 168 isolates were not antagonistic to Bradyrhizobium sp. strain NC-92 using dual culture technique. Further testing found 16 isolates of Bacillus spp. had the ability to inhibit mycelial growth of Rhizoctonia solani, a causal agent of leaf blight of bambara groundnut. Among these isolates, Bacillus spp. isolate TRV 9-5-2 had the greatest activity in anti-microbial tests against R. solani. This isolate was later identified as B. firmus. A powder formulation of B. firmus was developed by mixing bacterial endospores, talcum, sodium carboxymethylcellulose (SCMC) and polyvinylpyrolidone (PVP). The formulations contained bacterial levels ranging from 10⁸ to 10¹⁰ c.f.u./g and the viability of bacteria in all formulations remained high after 1 year storage at room temperature (26–32 °C). All formulations showed satisfactory effectiveness in vitro in suppressing mycelial growth of R. solani using dual culture technique. The application of formulations as seed treatment showed that these formulations did not cause abnormality of seedling shape and had no effect on the germination of bambara groundnut seeds.     

Industrial wastewaters and dewatered sludge: rich nutrient source for production and formulation of biocontrol agent, <Emphasis Type="Italic">Trichoderma viride</Emphasis>

Axenic cultivation of biocontrol fungus Trichoderma viride was conducted on a synthetic medium and different wastewaters and wastewater sludges in shake flasks to search for a suitable raw material resulting in higher biocontrol activity. Soluble starch based synthetic medium, dewatered municipal sludge, cheese industry wastewater sludge, pre-treated and untreated pulp and paper industry wastewater and slaughter house wastewater (SHW) were tested for T. viride conidia and protease enzyme production. The maximum conidia production followed the order, soluble starch medium (>10⁹ c.f.u./mL), untreated pulp and paper industry wastewater (4.9 × 10⁷ c.f.u./mL) > cheese industry wastewater (1.88 × 10⁷ c.f.u./mL) ≈ SHW (1.63 × 10⁷ c.f.u./mL) > dewatered municipal sludge (3.5 × 10⁶ c.f.u./mL) > pre-treated pulp and paper industry wastewater (1.55 × 10⁶ c.f.u./mL). The protease activity of T. viride was particularly higher in slaughterhouse wastewater (2.14 IU/mL) and dewatered municipal sludge (1.94 IU/mL). The entomotoxicity of soluble starch based synthetic medium was lower (≈6090 SBU/μL) in contrast to other raw materials. The entomotoxicity inversely decreased with carbon to nitrogen ratio in the growth medium and the conidia concentration and protease activity also contributed to the entomotoxicity. The residual c.f.u./g formulation of T. viride conidia were up to approximately, 90% after 1 month at 4 ± 1 °C and about 70% after 6 months at 25 ± 1 °C. Thus, production of T. viride conidia would help in marketability of low cost biopesticide from the sludge and safe reduction of pollution load.     

Submerged culture of a mycelial formulation of a bioherbicidal strain of <Emphasis Type="Italic">Myrothecium </Emphasis><Emphasis Type="Italic">verrucaria</Emphasis> with mitigated mycotoxin production

A mycelial formulation of the fungus Myrothecium verrucaria (IMI 361690) containing 0.20% Silwet L-77 surfactant was found to be highly efficacious in controlling the exotic invasive weed kudzu. The mycelium can be rapidly (48–72 h) produced in several media, including an inexpensive soy flour–corn meal medium. Mycelial yields were 2, 10, and 25 g dry weight l⁻¹ in Czapek-Dox, Richard’s V-8, and soy flour–corn meal media, respectively. Scale-up production in soy flour–corn meal medium using laboratory fermenters (10–25 l), resulted in a mycelial formulation that caused 90% mortality of naturally-occurring mature (0.9–1.0 m in height) kudzu within 48 h after application in field experiments. HPLC analyses revealed that the mycelium produced in this liquid culture contained no detectable amounts of the trichothecene mycotoxins roridin A and verrucarin A (limit of detection 2 μg ml⁻¹). This has resulted in a safer, yet effective bioherbicidal product. We anticipate that these findings should improve the probability of EPA registration and subsequent commercial development of this bioherbicide.     

Preparation and evaluation of Bacillus megaterium-alginate microcapsules for control of rice sheath blight disease

Bacillus megaterium encapsulated in calcium alginate microcapsules was prepared and tested for its efficacy against sheath blight disease of rice. In laboratory conditions, the aqueous suspension (1:100, v/v in potato dextrose agar) of the bacterial microcapsules (10¹⁰ spores/ml) inhibited mycelial growth of Rhizoctonia solani (>99 %) after the microcapsules were produced and stored for 12 months at room temperature (28 ± 2 °C). The survival of the bacterium in the microcapsules in response to ultraviolet (u.v.) irradiation and high temperature was investigated. The survivability of the bacterium in the encapsulated form was greater than that of the fresh cells when it was subjected to u.v. (20-W General electric u.v. lamp from a 25 cm distance for 48 h) and a high temperature treatment (80 °C for 48 h). Cells of the bacterium were detected by scanning electron microscope on both the leaf sheath and the leaf blade (in pot tests in a greenhouse) after spraying encapsulated product. The number of bacteria on the surface of both rice tissues (5 Log. number/g of plant) after spraying with encapsulated product was not significantly different from that after spraying with fresh cells onto the rice seedlings. Spraying the encapsulated B. megaterium on rice plants in the greenhouse was as effective as spraying a chemical fungicide for suppressing rice sheath blight disease.     

Measurement of particle diameter of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> microcapsule by spray drying and analysis on its microstructure

Lactobacillus acidophilus, as a probiotic, is widely used in many functional food products. Microencapsulation not only increases the survival rate of L. acidophilus during storage and extends the shelf-life of its products, but also optimal size microcapsule makes L. acidophilus have an excellent dispersability in final products. In this paper, L. acidophilus was microencapsulated using spray drying (inlet air temperature of 170°C; outlet air temperature of 85–90°C). The wall materials used in this study were β-cyclodextrin and acacia gum in the proportion of 9:1 (w/w), and microcapsules were prepared at four levels of wall materials (15, 20, 25 and 30% [w/v]) with a core material concentration of 6% (v/v). The microcapsule diameters were measured by Malvern’s Mastersizer-2000 particle size analyzer. The results showed that the particle diameters of microcapsule were mostly within 6.607 μm and 60.256 μm and varied with 2.884–120.226 μm (the standard smaller microcapsule designated as <350 μm). Through comparison of microcapsule size and uniformity with different concentration of wall materials, we concluded that the optimal concentration of wall material was 20% (w/v), which gave microcapsule with a relatively uniform size (averaging 22.153 μm), and the number of surviving encapsulated L. acidophilus was 1.50 × 10⁹ c.f.u./ml. After 8 weeks storage at 4°C, the live bacterial number was above 10⁷ c.f.u./ml, compared with unencapsulated L. acidophilus, 10⁴–10⁵ c.f.u./ml. Through the observation of scanning electron microscopy, we found that the shapes of microcapsule were round and oval, and L. acidophilus cells located in the centre of microcapsule.     

Control of blast and sheath blight diseases of rice using antifungal metabolites produced by Streptomyces sp. PM5

Two antifungal aliphatic compounds, SPM5C-1 and SPM5C-2 with a lactone and ketone carbonyl unit, respectively obtained from Streptomyces sp. PM5 were evaluated under in vitro and in vivo conditions against major rice pathogens, Pyricularia oryzae and Rhizoctonia solani. These compounds were dissolved in distilled water/medium to get the required concentrations. The well diffusion bioassay indicated that the of SPM5C-1 remarkably inhibited the mycelial growth of P. oryzae and R. solani in comparison to SPM5C-2. Though SPM5C-2 showed low antifungal activity against P. oryzae, it was not active against R. solani. Further, SPM5C-1 completely inhibited the growth of P. oryzae and R. solani at concentrations of 25, 50, 75 and 100 μg/ml. Greenhouse experiments revealed that spraying of SPM5C-1 at 500 μg/ml on rice significantly decreased blast and sheath blight development by 76.1% and 82.3%, respectively, as compared to the control with a corresponding increase in rice grain yield.     

Preparation of a flowable liquid bacterial insecticide based on Bacillus sphaericus

Fermenter-produced Bacillus sphaericus 2362 was formulated into a thick, dark flowable liquid concentrate containing 4.8×10⁹ c.f.u./ml and charcoal as protector against ultraviolet light. The potencies of the formulation against L₄ Culex pipiens quinquefasciatus before and after storage for 2 years were 5714 and 5862 International Toxic Units (ITU), respectively, when compared with a standardized B. sphaericus from the WHO at 1000 ITU. In field trials, treatment at 1.01/ha gave 96 to 100% control of mosquito larvae. B. sphaericus could be re-isolated in 5% of the samples 9 months after application.The authors are at the Department of Applied Microbiology & Brewing, Anambra State University of Technology, P.M.B. 5025, Awka, Nigeria.     

Endophytic bacterial flora in the stem tissue of a tropical maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) genotype: isolation,identification and enumeration

The genotypic diversity of indigenous bacterial endophytes within stem of tropical maize (Zea mays L.) was determined in field and greenhouse experiments. Strains were isolated from stem tissues of a tropical maize cultivar (PEHM-1) by trituration and surface disinfestation and their population dynamics was determined. Endophytes were found in most of the growing season at populations ranging from 1.36–6.12 × 10⁵ colony-forming units per gram fresh weight (c.f.u./gm fw) of stem. Analysis of these bacterial endophytes using Gas Chromatography—Fatty Acid Methyl Ester (GC-FAME) led to the identification of Bacillus pumilus, B. subtilis, Pseudomonas aeruginosa and P. fluorescens as the relatively more predominant group of bacterial species residing in maize stem. When the maize seedlings grown in a greenhouse were inoculated with these four isolates individually, their population densities decreased (1.6–3.1 × 10⁵ c.f.u./gm fw of stem) as compared to the field-grown maize (1.8–3.8 × 10⁵ c.f.u./gm fw of stem). The highest persistence, however, was recovered in the case of B. subtilis with a population density of 3.1 × 10⁵ c.f.u./gm fw of stem tissue on 28 days after emergence (DAE). This is the first report on population dynamics of bacterial endophytes from tropical maize and the results establish that symptomless populations of bacteria exist in the maize stem.     

Field persistence of the entomopathogenic nematode <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis> in different crops

To investigate nematode establishment and persistence, dauer juveniles (DJs) of Heterorhabditis bacteriophora were applied at 50 cm^-2 in different crops in June and July with conventional spraying equipment and 420 l water ha^-1. Application hardly had any effects on survival and infectivity. The number of DJs reaching the soil was assessed and the establishment and persistence recorded by baiting soil samples with larvae of the wax moth Galleria mellonella. The better the plant canopy was developed the fewer DJs reached the soil during application. Whereas in pasture 77% and in potatoes 78% of the applied nematodes reached the soil, in wheat and peas little less than 50%, in oil-seed rape only 5% and in lupine 6% were recorded. Between 50 and 60% of the soil samples contained H. bacteriophora a month after application with the exception of wheat (>90%) and potatoes (<5%) indicating that the number of nematodes reaching the soil during application had no influence on their establishment in the soil. Probably DJs can survive in the plant canopy and reach the soil later after application. The percentage of nematode-positive soil samples dropped considerably after tillage. In potatoes no nematodes were recovered after two months, which probably was also due to the intensive movement of the soil. Although nematodes are susceptible to freezing, temperatures below 0°C during the winter did not extinguish the H. bacteriophora population. In field crops EPN usually persisted not much longer than one year. The longest persistence of H. bacteriophora was detected 23 months after release in beans followed in rotation by wheat with red clover as cover crop. In this field larvae of the pea weevil Sitona lineatus (Coleoptera: Curculionidae) were detected in soil samples and found infected with the released nematode population. In the laboratory the field soils were tested for persistence of H. bacteriophora at 8°C and a half-life of 24.8 days was recorded in the absence of host insects and plants. Thus long-term persistence in the field was a result of recycling in host insects, which could not be detected in other crops than beans and clover. As H. bacteriophora seems to be restricted in its host potential, this species disappears after release once the host population is not available anymore.     

In vitro efficacy of <Emphasis Type="Italic">Hyptis suaveolens</Emphasis> L. (Poit.) essential oil on growth and morphogenesis of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">gladioli</Emphasis> (Massey) Snyder &; Hansen

Hyptis suaveolens L. (Poit.) essential oil was tested in vitro on the growth and morphogenesis of Fusarium oxysporum f.sp. gladioli (Massey) Snyder & Hansen, which causes Fusarium corm rot and yellows in various susceptible cultivars of gladiolus. The fungitoxicity of the oil was measured by percentage radial growth inhibition using the poisoned food technique (PF) and volatile activity assay (VA). The mycelial growth of the test fungus was completely inhibited at 0.998 and 0.748 μg ml⁻¹ concentration of oil in PF and VA, respectively. Essential oil was found to be fungicidal in nature at 1.247 and 0.998 μg ml⁻¹ concentration of oil in PF and VA, respectively. Determination of conidial germination in the presence of oil was also carried out and it was found that the oil exhibited 100% inhibition of conidial germination at 0.450 μg ml⁻¹ concentration. The effect of essential oil on the yield of mycelial weight was observed and it was found that at 0.873 μg ml⁻¹ concentration no mycelium was recorded and 100% inhibition was observed. The fungitoxicity of oil did not change even on exposure to 100°C temperature or to autoclaving, and the oil also retained its fungicidal nature even after storage of 24 months. The main changes observed under light microscopy after oil treatment were a decrease and loss of conidiation and anomalies in the hyphae such as a decrease in the diameter of hyphae and granulation of cytoplasm. The treatment of the oil also showed highly reduced cytoplasm in the hyphae, showing clear retraction of the cytoplasm from the hyphae and ultimately in some areas hyphae without cytoplasm were also found. GC-MS studies of the essential oil revealed that the oil consisted of 24 compounds with 1,8-cineole as major component accounting for 44.4% of the total constituents.     

Detection of living <Emphasis Type="Italic">Salmonella</Emphasis> cells using bioluminescence

ATP-based bioluminescence using mutant firefly luciferase was combined with an immunochromatographic lateral flow test strip assay for Salmonella enteritidis detection. In this combination method, the Salmonella-antibody–gold complex captured at the test line on the test strip was lysed by heat-treatment, and the ATP released from the cells was measured using mutant luciferase. This method resulted in approximately 1,000 times higher sensitivity in the detection of Salmonella (i.e. 10³ c.f.u./ml) compared to immunochromatographic lateral flow assay.     

Influence of nutritional conditions on exopolysaccharide production by submerged cultivation of the medicinal fungus <Emphasis Type="Italic">Shiraia bambusicola</Emphasis>

Maltose and yeast extract were the most favourable carbon and nitrogen sources for exopolysaccharide production by submerged culture of Shiraia bambusicola WZ-003, and initial maltose and yeast extract concentrations were at 30 and 3 g l⁻¹, respectively. Plant oils could increase the mycelial growth and exopolysaccharide production in tested concentration. K⁺ and Mg²⁺ could enhance the mycelial growth and exopolysaccharide biosynthesis. The optimal cultivation temperature and initial pH were found to be 26°C and 6.0, respectively. Exopolysaccharide concentration reached 0.53 g l⁻¹ in 15-l fermenter under optimal nutritional conditions.     

Use of plant extracts and biocontrol agents for the management of brown spot disease in rice

Fifty plant extracts, four oil cakes and eight antagonistic organisms were tested against Bipolaris oryzae (Cochliobolus miyabeanus), the causal agent of brown spot disease of rice. In vitro studies indicated that two leaf extracts, Nerium oleander and Pithecolobium dulce exerted the higher percent inhibition to mycelial growth (77.4, 75.1%) and spore germination (80.3, 80.0%) of B. oryzae. Among the four oil cake extracts tested in vitro against B. oryzae, neem cake extract showed the maximum inhibition percent to mycelial growth (80.18%) and spore germination (81.13%) of the pathogen followed by mahua cake extract, castor and gingelly cake extract. Trichoderma viride (Tv2) was significantly effective in inhibiting the mycelial growth (62.92%) and spore germination (77.03%) of the pathogen followed by Trichoderma harzianum (Th5) and Trichoderma reesei (Tr3). The promising leaf extracts, oil cake extracts and antagonistic microorganisms were further evaluated for their efficacies in disease management under glasshouse and field conditions. In glasshouse studies, post-infectional spraying of rice plants with neem cake extract, N. oleander leaf extract and T. viride (Tv2) was significantly effective in reducing the incidence of brown spot of rice by 66, 52 and 45 percent respectively. Two rounds of spraying of rice plants with neem cake extract, N. oleander leaf extract and T. viride (Tv2) in the field at initial appearance of disease and 15 days later reduced the incidence of brown spot (70, 53 and 48% disease reduction respectively) and increased the yield by 23, 18 and 15 percent respectively.     

Microbial population dynamics, especially stress tolerant Bacillus thuringiensis, in partially anaerobic rice field soils during post-harvest period of the Himalayan, island, brackish water and coastal habitats of India

Population ( × 10⁷ c.f.u./g dr. soil) of the aerobic (30.5–154.1) and anaerobic (5.9–91.4) heterotrophic, aerobic (24.0–56.0) and anaerobic (2.4–4.2) spore forming, Gram (-)ve (1.6–2.9), phosphate solubilizing (10–20), asymbiotic N₂-fixing (0.5–0.9), sulfur oxidizing (1.1–2.0), nitrifying (1.0–5.8) and denitrifying (12.1–18.7) bacteria; as well as, the actinomycetes (about 10⁴ c.f.u./g dr. soil) and fungi (about 10⁵ c.f.u./g dr. soil) were variable in the partially anaerobic, saline and drain out flooded rice soils during the post harvest period of the Himalayan, brackish water flooded, island and coastal habitats of India. The aerobic heterotrophic and spore forming bacteria were more than the anaerobic counterparts in the soils. Population (0.51–3.51 × 10⁶ c.f.u./g dr. soil) and crystal morphotype (spherical, bipyramidal and polymorphic) of the Bacillus thuringiensis (Bt) isolates of different soils were variable. Bt index was 0.002 at Mahe but 0.006 in other soils. The Bt isolates tolerated 5–12% NaCl. The osmolytes (mg/g dr. wt.) like the amino acids (0.38–99.45) and proline (0.38–0.80); and the antioxidative enzymes (units (U)/mg protein/min) viz. the catalase (0.17–5.59) and superoxide dismutase (0.35–74.46) were related with intrinsic osmotic stress tolerance of the Bt but they formed spores to overcome anoxic stress. Two Bt isolates were potent tolerant to both osmotic and anoxic stresses.     

Fluorescent pseudomonad mixtures mediate disease resistance in rice plants against sheath rot (<Emphasis Type="Italic">Sarocladium oryzae</Emphasis>) disease

Plant growth-promoting rhizobacterial (PGPR) strains were isolated from different agro-ecosystems of Tamil Nadu, India, and were tested for their efficacy against the sheath rot pathogen Sarocladium oryzae under in vitro, glasshouse and field conditions. Vigour and a relative performance index (RPI) were used to assay the growth promotion and antagonistic activity of Pseudomonas strains against S. oryzae under in vitro conditions. The results revealed the significant performance by strains Pf1, TDK1 and PY15 compared to other strains. Further, the combination of Pseudomonas strains Pf1, TDK1 and PY15 was more effective in reducing sheath rot disease in rice plants compared to individual strains under glasshouse and field conditions. Quantitative and native polyacrylamide gel electrophoresis (PAGE) analysis of peroxidase (PO), polyphenol oxidase (PPO) and chitinase activity in rice plants showed an increased accumulation of defence enzymes in the treatment with a combination of Pf1, TDK1 and PY15 compared to the treatment with individual strains and untreated controls. The present study revealed the probable influence of antagonism, plant growth promotion and induced systemic resistance (ISR) by the mixture of Pseudomonas bioformulations in enhancing the disease resistance in rice plants against sheath rot disease.

Conservation of urea-N by immobilization-remobilization in a rice-Azolla intercrop

Nitrogen losses are notoriously high in flooded rice fertilized with urea. An Azolla intercrop can reduce such losses by immobilizing urea-N during periods of potentially high N-loss. The reduction in N loss linked with the absorption and remobilization of urea-N by Azolla, was studied in two greenhouse experiments conducted in Goettingen (Germany). Grain yield and N recovery were positively influenced by Azolla more than doubling grain yield and N uptake as compared to the split application of 300 mg N pot^–1 alone (Exp. 1). In the second experiment, the yield increase was 78.3% with single applications of 97.5 and 68.4% after a split-application of a total of 195 mg N pot^–1. In both years the effect of urea and Azolla combined exceeded that of the sum of the factors alone, a clear positive synergistic effect on yield and N uptake by rice. Azolla effectively competed with the young rice plants for applied urea, capturing nearly twice the urea-N than the rice plants up to tillering in experiment 1. In the second experiment, 64.6 mg N of the 97.5 mg applied early in the season was immobilized by Azolla within 2 weeks. This represented 63.1% of the total N accumulated in the Azolla. The fraction of Azolla-N derived from urea sank to 36.4 mg within 4 weeks and only 27.2 mg at maximum tillering as a result of Azolla senescence and N-release. Of this 64.6 mg urea N immobilized 28.7% is eventually taken up by the standing rice plant, representing 43.1% of the remineralized, urea-derived Azolla N. Following the second urea application, only 17.9 mg N were immobilized in the Azolla biomass during the 2 weeks, of which 6.9 mg pot^–1 were still retained in the Azolla at maturity. At this stage, rice is the more effective competitor for applied N. As much as 42.1% of this immobilized N finds its way into the rice by maturity. Thus, Azolla contributed to the conservation of N in the system, particularly of the urea applied early in the season. Loss of N from the system amounted to no more than 15%. Although the early-applied N directly recovered by the rice plant was low (20%), 2/3 of the N captured by Azolla following this first urea application was released to the system by the time of rice harvest, over 40% of which was available to the rice plant. Azolla thus appears to act as a slow release fertilizer.     

Optimization of conditions for transient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated gene expression assays in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The β-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85–90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.     

Combined field efficacy of <Emphasis Type="Italic">Paranosema locustae</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var. <Emphasis Type="Italic">acridum</Emphasis> for the control of sahelian grasshoppers

Field trials were conducted to evaluate the efficacy of wheat bran bait formulations of Paranosema locustae and Metarhizium anisopliae for controlling grasshoppers in southeast Niger. Treatments consisted of wheat bran baits mixed with M. anisopliae, P. locustae + M. anisopliae or with P. locustae spores and P. locustae + sugar. Oedaleus senegalensis, Pyrgomorpha cognata and Acrotylus blondeli were the predominant species at the time of application representing ca. 94% of the total population. Bran application was done when O. senegalensis (ca. 75% of the population) was at its early developmental stages, with first, second and third instars accounting for 64–85%. Grasshopper population reduction, P. locustae prevalence and level of infections in the predominant species were monitored. Manual application of P. locustae and M. anisopliae formulated in wheat bran has proven to induce consistent pathogen infection in grasshopper populations. Population density over the three weeks monitoring, typically decreased by 44.7 ± 6.9%, 52.8 ± 8.4%, 73.7 ± 5.5% and 89.1 ± 1.8% in P. locustae, P. locustae + sugar, M. anisopliae and P. locustae + M. anisopliae treated plots respectively. Paranosema locustae prevalence in surviving adult grasshoppers at 28 after application was 48.1 ± 2.3%, 28.9 ± 4.8% and 27.4 ± 3.7%, with infection level of 6.2 ± 0.8 × 10⁶, 2.3 ± 0.3 × 10⁴ and 2.1 ± 0.3 × 10³ spores mg⁻¹ host weight in O. senegalensis, A blondeli and P. cognate respectively. Other species that each accounted for <2% of the community, namely Aiolopus thalassinus, A. simulatrix, Acorypha glaucopsis, Acrotylus patruelis, Anacridium melanorhodon, Diabolocatantops axillaris, Kraussaria angulifera and Schistocerca gregaria were found to show sign of infection. The results from this study suggest that wheat bran application of M. anisopliae and P. locustae alone or in combination, targeting early instars grasshopper could be a valuable option in grasshopper control programs.     

Tumor-inhibitory and liver-protective effects of <Emphasis Type="Italic">Phellinus igniarius</Emphasis> extracellular polysaccharides

The tumor-inhibitory and liver-protective effects of crude extracellular polysaccharides (EPS) extracted from the liquid mycelial culture of the mushroom Phellinus igniarius were studied in mice. The mice were injected with murine sarcoma S180 and murine hepatoma H22. Crude EPS at 100, 200, 400 mg kg⁻¹ body weight was administered to EPS groups each day in the twelve consecutive days. The result showed that EPS 200 mg kg⁻¹ body weight significantly inhibited S180 and H22 at 65.0 and 46.3%, respectively. Moreover, EPS could not only keep the numbers of WBC, RBC, PLT and the concentration of HGB in a normal range, but also normalize the activities of AST, ALT and ALP. For example, in EPS-treated mice, AST significantly reduced with the percentage of A/G reverse in S180 (P < 0.05) and H22 (P < 0.01) when the mice took EPS 200 mg kg⁻¹ body weight. In conclusion, it was remarkable that P. igniarius EPS exhibited antitumor activity related to dosage and protected liver function by sustaining the blood routine as well as keeping the blood biochemical indexes normal.