Incidence of aflatoxins and aflatoxin producing fungi in animal feedstuffs

Fifty four samples including 5 of broken rice, 8 of corn grains, 8 of corn gluten feed, 13 of cottonseed cake and 4 each of rice polish, corn gluten, sesame oil cake, guar meal and wheat bran were screened for the presence of aflatoxins. Among all the samples, 14 were damaged and 40 apparently undamaged. The incidende of aflatoxins was found to be 60, 25, 25, and 23 per cent in broken rice, corn grains, corn gluten feed and cottonseed cake. Aflatoxins were not detected from rice polish, corn gluten, sesame oil cake, guar meal and wheat bran. Damaged sample revealed a much higher incidence i.e. 50 per cent as compared to undamaged ones i.e. 7.5 per cent. Mean concentration of aflatoxin B and G was found to be 15.5 and 12.2 ppb respectively.Cultural examination of aflatoxin positive feedstuffs yielded 39 isolates of different fungi including 21 of Aspergillus, 7 of Mucor, 6 of Rhizopus, 4 of Fusarium and one of Penicillium. These strains when tested for aflatoxin producing ability, revealed this property in only one isolate, identified as Aspergillus parasiticus.     

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.     

Changes in moisture content,mycoflora and aflatoxin content of rice bran during storage

The changes in moisture content, storage mycoflora and aflatoxin B₁ (AFB₁) in bran from untreated or raw rice (Rr) and parboiled rice (Pbr) stored in small lots in polyethylene bags were studied at 15-day intervals up to 60 days, using five lots of each type of bran. Deterioration was more rapid with reference to all the three parameters, in Rr bran compared to Pbr bran, the former becoming completely overgrown and caked with fungi by the end of 60 days.Aspergillus flavus was the dominant fungus in Pbr bran, whereasA. candidus andTrichoderma viride were abundant in Rr bran. The frequency of incidence as well as concentration of AFB₁ increased with storage time in both types of bran, but the rate of increase as well as overall concentration were much higher in Rr bran. Thus raw rice bran is unsuitable for prolonged storage.Abbreviations AFB₁ aflatoxin B₁ - MC moisture content - Pbr parboiled rice - Rr raw rice     

Production of high activity thermostable phytase from thermotolerant Aspergillus niger in solid state fermentation

The thermotolerant fungus, Aspergillus niger NCIM 563, was used for production of extracellular phytase on agricultural residues: wheat bran, mustard cake, cowpea meal, groundnut cake, coconut cake, cotton cake and black bean flour in solid state fermentation (SSF). Maximum enzyme activity (108 U g⁻¹ dry mouldy bran, DMB) was obtained with cowpea meal. During the fermentation phytic acid was hydrolysed completely with a corresponding increase in biomass and phytase activity within 7 days. Phosphate in the form of KH₂PO₄ (10 mg per 100 g of agriculture residue) increased phytase activity. Among various surfactants added to SSF, Trition X-100 (0.5%) exhibited a 30% increase in phytase activity. The optimum pH and temperature of the crude enzyme were 5.0 and 50°C respectively. Phytase activity (86%) was retained in buffer of pH 3.5 for 24 h. The enzyme retained 75% of its activity on incubation at 55°C for 1 h. In the presence of 1 mM K⁺ and Zn²⁺, 95% and 55% of the activity were retained. Scanning electron microscopy showed a high density growth of fungal mycelia on wheat bran particles during SSF. Journal of Industrial Microbiology & Biotechnology (2000) 24, 237–243. Received 07 June 1999/ Accepted in revised form 18 December 1999     

L-glutaminase production by <Emphasis Type="Italic">Trichoderma koningii</Emphasis> under solid-state fermentation

Solid state fermentation was conducted for the production of L-glutaminase by Trichoderma koningii Oud.aggr. using different agro-industrial byproducts inlcuding wheat bran, groundnut residues, rice hulls, soya bean meal, corn steep, sesamum oil cake, cotton seed residues and lentil industrial residues as solid substrates. Wheat bran was the best substrate for induction of L-glutaminase (12.1 U/mg protein) by T. koningii. The maximum productivity (23.2 U/mg protein) and yield (45.0 U/gds) of L-glutaminase by T. koningii occurred using wheat bran of 70% initial moisture content, initial pH 7.0, supplemented with D-glucose (1.0%) and L-glutamine (2.0% w/v), inoculated with 3 ml of 6 day old fungal culture and incubated at 30°C for 7 days. After optimization, the productivity of L-glutaminase by the solid cultures of T. koningii was increased by 2.2 fold regarding to the submerged culture.     

Exploitation of inexpensive substrates for production of a novel SCL–LCL-PHA co-polymer by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> MTCC 7925

Studies conducted with various inexpensive carbon sources such as whey, vegetable oils (palm, mustard, soybean and coconut), a low-cost source of glucose-D, rice and wheat bran, and mustard and palm oil cakes demonstrated palm oil as the best substrate for accumulation of a novel short-chain-length–long-chain-length polyhydroxyalkanoate (SCL–LCL-PHA) co-polymer containing SCL 3HAs [3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV)] and LCL 3HAs of 3-hydroxyhexadecanoic acid (3HHD) and 3-hydroxyoctadecanoic acid (3HOD) units as constituents by a sludge-isolated Pseudomonas aeruginosa MTCC 7925. The co-polymer content reached up to 60% of dry cell weight (dcw) at 48 h of incubation in 0.5% (v/v) palm oil and the extract of 0.5% (v/v) palm oil cake supplemented vessels. The PHAs pool was further enhanced up to 69 and 75% (dcw), when the above culture was subjected to P- and N-limitation, respectively. The mol fraction of 3HB:3HV:3HHD:3HOD units were, respectively, 83.1:7.7:3.8:5.4 and 87.3:5.1:3.6:4.0 in P- and N-limited cultures. Consequently, a co-polymer yield of 5 g l⁻¹ (approx.) was achieved, which was about 80-fold higher as compared to 69 mg l⁻¹ of the control culture. On substrate basis, the accumulation reached up to 0.62 g PHAs per g substrate, which was significantly higher as compared to the yield obtained from starch by Haloferax mediterranei and Azotobacter chroococum, from molasses by A. vinelandii UWD, and from lactose and xylose by Pseudomonas cepacia. This novel P(3HB-co-3HV-co-3HHD-co-3HOD) co-polymer exhibited better thermal and mechanical properties as revealed from the differential scanning calorimetry and mechanical property studies, thus opens up new possibilities for various industrial applications.     

Effectiveness of beta-exotoxin ofBacillus thuringiensis var.thuringiensis on the ability ofMeloidogyne sp. from brinjal (Solanum melongena L.) to survive

Beta-exotoxin produced byBacillus thuringiensis var.thuringiensis grown in the acid hydrolysates of wheat and rice brans caused 95% and 85% mortality respectively ofMeloidogyne sp. as against 72% of β-exotoxin produced on farm yard manure within 7 days. Acid hydrolysate of wheat or rice bran and solid farm yard manure proved to be the best media for growth ofB. thuringiensis var.thuringiensis.     

Kinetics of Enhanced Substrate Consumption and Endo-β-xylanase Production by a Mutant Derivative of Humicola lanuginosa in Solid-state Fermentation

Summary Industrial byproducts namely canola meal, rice bran, sunflower meal, and wheat straw were used as substrates for endo-xylanase production by Humicola lanuginosemutant TH1 through solid substrate fermentation. The enzyme was secreted extracellularly by both wild and mutant cultures. Rice bran supported the maximum production of endo-xylanase followed by wheat straw, canola meal and sunflower meal. The highest activity was achieved after 72 h of culture and the highest yields from the above substrates were 842, 840, 610 and 608 IU per g substrate consumed respectively. The highest productivity (281 IU flask⁻¹ h⁻¹ corresponding to 5620 l⁻¹ h^-1) of endo-xylanase by the mutant of H. lanuginosa was 1.6-fold more than that produced by the parental organism in solid-state fermentation of rice bran at 45 °C. Maximum specific activity (180 IU mg⁻¹ protein) and substrate consumption rates were significantly more than those reported by previous researchers on Humicola sp. The mutant possessed markedly low accompanying cellulase activity. Thermodynamic studies revealed that the mutant required significantly lower activation energy for enzyme production and higher for thermal inactivation which signified that the endogenous metabolic machinery of mutant cells exerted more protection against thermal inactivation during product formation than that needed by its parental cultures.     

Production of mosquitocidal <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> by solid state fermentation using agricultural wastes

In this study, Bacillus sphaericus NRC 69 was grown in culture media, in which 12 agricultural wastes were tested as the main carbon, nitrogen and energy sources under solid state fermentation. Of the 12 tested agricultural by-products, wheat bran was the most efficient substrate for the production of B. sphaericus mosquitocidal toxins against larvae of Culex pipiens (LC₅₀ 1.2 ppm). Mixtures of tested agricultural wastes separately with wheat bran enhanced the produced toxicity several folds and decreased LC₅₀ between 3.7- and 50-fold in comparison with that of agricultural wastes without mixing. The toxicity of B. sphaericus grown in wheat bran/rice hull at 8/2 (g/g) and wheat bran/barley straw at 1/4 (g/g) showed the same toxicity as that in wheat bran medium (LC₅₀ decreased 17- and 16-fold, in comparison with that in rice hull or barely straw media, respectively). In wheat bran medium, the maximum toxicity of the tested organism obtained at 50% moisture content, inoculum size 84 × 10⁶ CFU/g wheat bran and incubation for 6 days at 30°C. Addition of cheese whey permeate at 10% to wheat bran medium enhanced the toxicity of B. sphaericus NRC 69 about 46%.     

Protease production by Rhizopus oligosporus in solid-state fermentation

Rice bran was superior to other proteinaceous substrates for protease production by Rhizopus oligosporus ACM 145F in solid-state fermentation. Maximum protease yield was after 72 h. The optimal initial moisture content was 47% (a _w=0.97). Dried, ground and resuspended fermented rice was the most pratical and effective inoculum preparation, although, in the laboratory, spore suspensions prepared directly from agar slants were more convenient. Inoculum density (from 10² to 10⁷ spores/g substrate) and age (3, 5, 7 and 9 days) had little effect on protease yield.The authors are with the Department of Chemical Engineering, The University of Queensland, Brisbane, Queensland, 4072, Australia     

Protein enrichment and digestibility of soft rush (<Emphasis Type="Italic">Juncus effusus</Emphasis>) and rice residues using edible mushrooms <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> and <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis>

Pleurotus species are found to be among the most efficient lignocellulolytic types of white-rot fungi. Rice is the main grain cultivated in the extreme south of Brazil. Defatted rice bran and straw are by-products of low aggregate value. Soft rush (Juncus effusus) is a common native plant also very abundant in the region. In the present work, we evaluated changes in substrate composition after growth of two white-rot fungal species: Pleurotus ostreatus and Pleurotus sajor-caju, aiming to increase protein content and digestibility from substrates through solid fermentations and obtain edible mushrooms of high aggregate value. For that, defatted rice bran, defatted rice straw and soft rush were utilized as substrate. The influence of the variables thermal treatment temperature of substrate, substrate moisture and concentration were evaluated on the protein content, digestibility and biological efficiency. The highest protein enrichment of rice bran in P. sajor-caju-fermented medium was due the fact that there was no fructification in these media, while for the P. ostreatus-fermented medium, part of the synthesized protein was converted into mushrooms. The highest protein enrichments were verified in medium with 80% moisture and 25% soft rush (47.1% using P. ostreatus and 49.0% using P. sajor-caju). A higher digestible protein increase was obtained for both species in media with 70% moisture and 25% soft rush.     

Effect of plant oils on quality parameters of Bactrocera tryoni (Froggatt) reared on liquid larval diet

Liquid larval diets have been developed for several tephritid fruit flies including Queensland fruit fly, Bactrocera tryoni (Frogatt) (Q‐fly). In liquid diets, wheat germ oil (WGO) is usually added to improve performance in some quality parameters of reared flies, especially flight ability. However, for some flies, other plant oils may be more readily available, cheaper or produce flies of superior performance. In the present study, four alternative types of plant oils – rice bran, canola, vegetable, and sesame – were incorporated into a fruit fly liquid larval diet to replace the currently used wheat germ oil and their efficacy on the quality parameters of reared Q‐fly was compared to diets containing wheat germ oil or no oil. The quality parameters included: total pupal yield (N), pupal recovery (%), larval duration (days), pupal weight (mg), adult emergence (%), adult fliers (%), rate of fliers (%), sex ratio (%), F₁ egg/female/day and egg hatching (%). There were significant differences among treatments in performance of Q‐fly. Vegetable oil appeared better in terms of total pupal yield, percentage of pupal recovery, percentage of adult emergence, percentage of fliers, mean egg/female/day and % F₁ egg hatch compared with other oil treatments, especially from that of WGO treated diet. The result suggests that WGO can be substituted with rice bran and vegetable oil to improve the liquid larval diet for rearing of B. tryoni, with vegetable oil being the best replacement.     

Nitrogen-15 balances for urea and neem coated urea applied to lowland rice following two cowpea cropping systems

Little is known about whether the high N losses from inorganic N fertilizers applied to lowland rice (Oryza sativa L.) are affected by the combined use of either legume green manure or residue with N fertilizers. Field experiments were conducted in 1986 and 1987 on an Andaqueptic Haplaquoll in the Philippines to determine the effect of cowpea [Vigna unguiculata (L.) Walp.] cropping systems before rice on the fate and use efficiency of¹⁵N-labeled, urea and neem cake (Azadirachta indica Juss.) coated urea (NCU) applied to the subsequent transplanted lowland rice crop. The pre-rice cropping systems were fallow, cowpea incorporated at the flowering stage as a green manure, and cowpea grown to maturity with subsequent incorporation of residue remaining after grain and pod removal. The incorporated green manure contained 70 and 67 kg N ha⁻¹ in 1986 and 1987, respectively. The incorporated residue contained 54 and 49 kg N ha⁻¹ in 1986 and 1987, respectively. The unrecovered¹⁵N in the¹⁵N balances for 58 kg N ha⁻¹ applied as urea or NCU ranged from 23 to 34% but was not affected by pre-rice cropping system. The partial pressure of ammoniapNH₃, and floodwater (nitrate + nitrite)-N following application of 29 kg N ha⁻¹ as urea or NCU to 0.05-m-deep floodwater at 14 days after transplanting was not affected by pre-rice cropping system. In plots not fertilized with urea or NCU, green manure contributed an extra 12 and 26 kg N ha⁻¹, to mature rice plants in 1986 and 1987, respectively. The corresponding contributions from residue were 19 and 23 kg N ha⁻¹, respectively. Coating urea with 0.2g neem cake per g urea had no effect on loss of urea-N in either year; however, it significantly increased grain yield (0.4 Mg ha⁻¹) and total plant N (11 kg ha⁻¹) in 1987 but not in 1986.     

Behavioural and population responses to changing availability of <Emphasis Type="Italic">Artemia</Emphasis> prey by moulting black-necked grebes, <Emphasis Type="Italic">Podiceps nigricollis</Emphasis>

Chemical communication may inform about the location of prey, predators, co-specifics, and mate partners in zooplankton. In this study, we evaluated several life-history traits of the rotifer, Brachionus calyciflorus, exposed to conditioned media by a rotifer predator (Asplanchna brightwelli) and a cladocera competitor (Daphnia similis), quantifying population growth and life-table demography at two algal food levels (2.0 and 0.5 × 10⁶ cells ml⁻¹ of Chlorella pyrenoidosa). At both food levels, B. calyciflorus grown in predator-conditioned media had lower population abundance and slower population growth rate than controls. Conversely, the competitor-conditioned media treatments produced both higher rotifer population abundance and faster population growth rate than controls. Life-history parameters varied significantly depending on the presence of predator and competitor-conditioned media. The Asplanchna-conditioned media significantly decreased gross reproductive rate (GRR): 8–9 offsprings per female; net reproductive rate (R ₀): 6–7 offsprings per female; population growth rate (r): 0.34–0.37 day⁻¹; and increased generation time (T): 5.5–5.6 days. On the other hand, The Daphnia-conditioned media significantly increased the GRR (13–14 offsprings per female); net reproductive rate (8–9 offsprings per female); population growth rate (0.42–0.43 day⁻¹); and decreased generation time (4.9–5.0 days). However, the effects of food level on the life-history characteristic were not significant in both treatments. Maximum values of the population abundance and the population growth rate are significantly influenced by the predator densities and pre-culture time. This study suggests that rotifers use variable life-history strategies (low reproduction and high survivorship versus high reproduction and low survivorship) based on the presence of predators and competitors.     

Evaluation of shelf life and rock phosphate solubilization of Burkholderia sp. in nutrient-amended clay, rice bran and rock phosphate-based granular formulation

Five phosphate-solubilizing bacteria (PSB) used in this study were isolated based on their ability to solubilize tricalcium phosphate (TCP) in Pikovskaya’s medium. Among the tested bacterial strains Burkholderia sp. strain CBPB-HIM showed the highest solubilization (363 μg of soluble P ml⁻¹) activity at 48 h of incubation. Further, this strain has been selected to assess its shelf life in nutrient-amended and -unamended clay, rice bran and rock phosphate (RP) pellet-based granular formulation. The results showed that the maximum viability of bacterium was observed in clay and rice bran (1:1) + 10% RP pellets than clay-RP pellets, irrespective of tested storage temperatures. Further, clay and rice bran (1:1) + 10% RP pellets amended with 1% glucose supported the higher number of cells compared to glycerol-amended and nutrient-unamended pellets. In this carrier solubilization of Morocco rock phosphate (MRP) by Burkholderia sp. strain CBPB-HIM was also investigated. The maximum of water and bicarbonate extractable P (206 and 245 μg P g⁻¹ of pellet respectively) was recorded in clay and rice bran (1:1) + 10% RP pellets amended with 1% glucose and glycerol respectively on day 5 of incubation. Therefore, this study proved the possibility of developing granular inoculant technology combining clay, rice bran and RP as substrates with phosphate-solubilizing Burkholderia.     

Natural occurrence of toxigenic fungi and mycotoxins in rice bran

Thirty four samples of rice bran, of which 9 were from raw (untreated) rice (RR) and 25 from parboiled rice (PbR) were collected from commercial rice mills in and around Madras and analysed for storage mycoflora and mycotoxins. Fungi of the Aspergillus flavus group were present in 29 of the 34 samples (8 from RR and 21 from PbR) in quantities ranging from <1–432 thousand propagules/g, though not always as the dominant mycoflora. Fungal numbers were usually higher in RR than in PbR samples.Five of the 9 RR samples and 6 of the 25 PbR samples were positive for aflatoxins. Among 29 isolates of A. flavus obtained from the bran samples, 16 isolates –6 from RR bran and 10 from PbR bran — were found to be toxigenic in vitro. Some isolates of A. candidus also seemed to produce aflatoxin and other fluorescent substances.     

Population structure and reproductive biology of Atlantoscia floridana (van Name, 1940) (Crustacea, Isopoda, Oniscidea) in southern Brazil

Data were obtained on the population structure and reproduction of Atlantoscia floridana, one of the most common species of terrestrial isopods in the restinga (coastal dune) forests of the state of Rio Grande do Sul, southern Brazil. During a 19-month period, a total of 7833 individuals were sampled: 2792 males, 3400 females and 1691 mancas. There was a significant difference between the size of both males and females collected in 2000 and 2001: the mean size was smaller in the second year when individuals in the larger size classes were lacking. Population density varied with season. The minimum population was 131 ind per m² individuals, the maximum 1040 ind per m² and the mean 450  per m². While the overall sex ratio was clearly female biased, the operational sex ratio favored males, and showed no changes with season. Because both ovigerous and post-ovigerous females were present throughout the year, reproduction is considered continuous; however, reproduction peaked during autumn and spring. Ovigerous females were measured (CW = cephalothorax width) and the number of eggs was counted. Fecundity (F) varied from 5 to 23 eggs ( = 11.18 ± 4) per female, and was expressed by the regression F = –18.48 + 22.59 CW, with the female cephalothorax width varying from 1.04 to 1.68 mm. Marsupial mortality was only 0.9%. Egg production was 588 eggs per m² in spring and 660 eggs per m² in autumn. Recruitment occurred in all months, and eggs, embryos and marsupial mancas were also present year-round. A. floridana is the dominant species of terrestrial isopod in the study area. Its most remarkable characteristic is its high reproductive investment.     

Effect of Temperature on the Life History of Cirrospilus sp. near lyncus (Hymenoptera: Eulophidae), a Parasitoid of Phyllocnistis citrella (Lepidoptera: Gracillariidae)

The effect of four constant temperatures on the life history of Cirrospilus sp. near lyncus was examined in the laboratory. This species is one of the most abundant generalist indigenous parasitoids of the citrus leafminer, Phyllocnistis citrella Stainton, in Spain. Adult lifespan of C. sp. near lyncus decreased from 50.2 to 9.1 days as temperatures increased from 15 to 30°C, respectively. Both gross fecundity and host-feeding were highest at 20°C (170.48 eggs and 32.33 hosts). Oviposition rates were optimal at higher temperatures (5.22 eggs per day at 25°C and 4.79 eggs per day at 30°C) and were dependent on female age. In contrast, host-feeding rates for a given temperature did not depend on age. Generation time decreased with increasing temperatures from 68.05 days at 15°C to 12.19 days at 30°C. Net reproduction peaked at 20°C (68.86 viable females per female). Intrinsic rate of increase doubled from 15°C (0.059 females per female per day) to 20°C (0.127 females per female per day) and almost doubled again from 20 to 30°C (0.210 females per female per day). Given these parameters, C. sp. near lyncus could perform optimally in the area occupied by P. citrella in the Mediterranean region.     

Biological Control of Rice Blast byPseudomonas fluorescensStrainPf7–14: Evaluation of a Marker Gene and Formulations

Pseudomonas fluorescensstrainPf7–14 was evaluated for biological control of rice blast in field experiments. StrainPf7–14 was formulated in methylcellulose:talc (1:4) and applied to IR50 rice (Oryza sativa) seeds as a seed treatment and as foliar sprays in seedbed and field experiments. When applied as a seed treatment followed by three foliar applications, the strain provided a 68.5% suppression of rice blast in the seedbed experiment and a 59.6% suppression in the field experiment. The persistence and migration ofPf7–14 on the rice plant was studied with the aid oflacZYgenes inserted into the bacterium. In greenhouse experiments,Pf7–14gal was detected on rice roots at 10⁶to 10⁵cfu/g of root tissue for 110 days, the duration of the rice crop. Migration of the strain from the seeds to the leaves occurred only until the seedlings were 16 days old. WhenPf7–14 was applied to the rice plants by foliar sprays, 10⁴cfu of the bacterium per gram of leaf tissue was detected for the next 40 days. The limited migration of the bacterial biocontrol agent emphasizes the need for multiple foliar applications of the bacterium to sustain the bacterial population for effective suppression of rice blast.