Conidia production by Isaria fumosorosea on solid substrates and its pathogenicity towards Bemisia tabaci

Large amounts of conidia must be recovered to use Isaria fumosorosea as a biological control agent. Aerial conidia are mass-produced in solid substrate culture. Conidia obtained from growth on different substrates must be similar in quantity and quality (viability, purity and virulence). In the present work, several solid substrates were evaluated: rice, sorghum, sugarcane, corncob, plantain and wheat (derived from the production of Trichogramma sp.) to find alternatives that are economical and available in different regions. The highest conidia production was obtained on rice, plantain and corncob substrates, which yielded 5.33 × 10⁹ conidia/g, 5.24 × 10⁹ conidia/g and 4.8 × 10⁹ conidia/g substrate, respectively. At concentrations of 1 × 10⁷ conidia/mL, the mortality rates obtained with conidia developed on rice, plantain and corncob substrates were 98%, 97% and 88%, respectively. The lethal concentration 50 (LC₅₀₎ values of conidia obtained from rice and plantain were 7.2 × 10⁴ conidia/mL and 5.1 × 10⁴ conidia/mL, respectively.     

Towards the production of fungal biocontrol candidates using inert supports: a case of study of Trichoderma asperellum in a pilot fixed bed fermenter

The production of fungal biocontrol agents by solid-state fermentation (SSF) processes in inert supports demands deeper studies in SSF-modelling and SSF-optimisation to cope with scale-up issues. Here, we report the systematic application of fractional factorial and central composite designs to optimise the conidia productivity and maximum specific growth rate of the biological control candidate Trichoderma asperellum strain Th204 using two inert supports: polyurethane foam (PUF) and rice husk (RH), in a pilot 16 L fixed bed fermenter. By using response surface methodology, 2D contour graphs and Spearman’s correlation coefficients, axial temperature, conidia concentration, bed moisture and pressure drop gradients were modelled. C:N ratio and airflow rate were identified as significant factors. Optimal conditions using PUF a C:N ratio of 18.1 and airflow of 0.8?m³?h^?1 were found, with the highest productivity of 3.09?×?10⁷conidia g^?1 initial dry matter h¹. Polynomial models and response surfaces found in this study are advantageous to design strategies to scale-up the SSF process in fixed bed fermenters for fungal biological control candidates.     

Studies on Exo-Chitinase Production from Trichoderma asperellum UTP-16 and Its Characterization

The growth conditions for chitinase production by Trichoderma asperellum UTP-16 in solid state fermentation was optimized using response surface methodology based on central composite design. The chitinase production was optimized, using one-factor at a time approach, with six independent variables (temperature, pH, NaCl, incubation period, nitrogen and carbon sources) and 3.31 Units per gram dry substrate (U gds⁻¹) exo-chitinase yield was obtained. A 21.15% increase was recorded in chitinase activity (4.01 U gds⁻¹) through surface response methodology, indicates that it is a powerful and rapid tool for optimization of physical and nutritional variables. Further, efficiency of crude enzyme was evaluated against phytopathogenic Fusarium spp. and a mycelial growth inhibition up to 3.5–6.5 mm was achieved in well diffusion assay. These results could be supplemented as basic information for the development of enzyme based formulation of T. asperellum UTP-16 and its use as a biocontrol agent.     

Production of cephamycin C by <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> NT4 using solid-state fermentation

Cephamycin C is an extracellular broad spectrum β-lactam antibiotic produced by Streptomyces clavuligerus, S. cattleya and Nocardia lactamdurans. In the present study, different substrates for solid-state fermentation were screened for maximum cephamycin C production by S. clavuligerus NT4. The fermentation parameters such as substrate concentration, moisture content, potassium dihydrogen phosphate, inoculum size and ammonium oxalate were optimized by response surface methodology (RSM). The optimized conditions yielded 21.68 ± 0.76 mg gds⁻¹ of cephamycin C as compared to 10.50 ± 1.04 mg gds⁻¹ before optimization. Effect of various amino acids on cephamycin C production was further studied by using RSM, which resulted in increased yield of 27.41 ± 0.65 mg gds⁻¹.     

Physiological and antioxidant response by Beauveria bassiana Bals (Vuill.) to different oxygen concentrations

The effect of three levels of oxygen (normal atmosphere (21% O₂), low oxygen (16% O₂) and enriched oxygen (26% O₂)) on the production and germination of conidia by Beauveria bassiana was evaluated using rice as a substrate. The maximum yield of conidia was achieved under hypoxia (16% O₂) after 8 days of culture (1.51 × 10⁹ conidia per gram of initial dry substrate), representing an increase of 32% compared to the normal atmosphere. However, germination was reduced by at least 27% due to atmospheric modifications. Comparison of antioxidant enzyme activity (superoxide dismutases and catalases) with the oxidation profiles of biomolecules (proteins and lipids) showed that a decrease in catalase activity in the final days of culture coincided with an increase in the amount of oxidized lipids, showing that oxidative stress was a consequence of pulses of different concentrations of O₂. This is the first study describing oxidative stress induction by atmospheric modification, with practical implications for conidia production.     

New Solid-State Fermentation Chamber for Bulk Production of Aerial Conidia of Fungal Biocontrol Agents on Rice

A novel solid-state fermentation apparatus, namely an upright multi-tray conidiation chamber, was developed to facilitate the production of aerial conidia of fungal biocontrol agents, such as Beauveria bassiana. The chamber with 25 bottom-meshed metal trays had a capacity of ≥50 kg rice with each tray holding ≥2 kg. In repeated trials, a mean yield of 2.4 (1.8–2.7) × 10¹² conidia kg⁻¹ rice was harvested from the 7-day cultures of B. bassiana in a fully loaded chamber. The new apparatus has a high potential for bulk production of fungal conidia.     

Production of microsclerotia by Brazilian strains of Metarhizium spp. using submerged liquid culture fermentation

We investigated the potential production and desiccation tolerance of microsclerotia (MS) by Brazilian strains of Metarhizium anisopliae (Ma), M. acridum (Mc) and M. robertsii (Mr). These fungi were grown in a liquid medium containing 16 g carbon l^?1 with a carbon:nitrogen ratio of 50:1. One hundred milliliters cultures were grown in 250 ml Erlenmeyer flasks in a rotary incubator shaker at 28 °C and 200 rpm for 5 days. Five-day-old MS were harvested, mixed with diatomaceous earth (DE) and air-dried for 2 days at 30 °C. The air-dried MS–DE granular preparations were milled by mortar + pestle and stored in centrifuged tubes at either 26 or ?20 °C. Desiccation tolerance and conidia production were assessed for dried MS granules by measuring hyphal germination after incubation for 2 days on water agar plates at 26 °C and for conidia production following 7 days incubation. Yields of MS by all strains of Metarhizium were 6.1–7.3 × 10⁶ l^?1 after 3 days growth with maximum MS yields (0.7–1.1 × 10⁷ l^?1) after 5 days growth. No differences in biomass accumulation were observed after 3 days growth, whereas Ma-CG168 showed the highest biomass accumulation after 5 days growth. Dried MS–DE preparations of all fungal strains were equally tolerant to desiccation (≥93 % germination) and the highest conidia production was obtained by MS granules of Mc-CG423 (4 × 10⁹ conidia g^?1). All MS granules showed similar stability after storage at either 26 or ?20 °C for 3.5 months.     

Selection of promising fungal biological control agent of the western flower thrips Frankliniella occidentalis (Pergande)

Aims: Larval stages of Frankliniella occidentalis are known to be refractory to fungal infection compared with the adult stage. The objective of this study was to identify promising fungal isolate(s) for the control of larval stages of F. occidentalis. Methods and Results: Ten isolates of Metarhizium anisopliae and eight of Beauveria bassiana were screened for virulence against second‐instar larvae of F. occidentalis. Conidial production and genetic polymorphism were also investigated. Metarhizium anisopliae isolates ICIPE 7, ICIPE 20, ICIPE 69 and ICIPE 665 had the shortest LT₅₀ values of 8·0–8·9 days. ICIPE 69, ICIPE 7 and ICIPE 20 had the lowest LC₅₀ values of 1·1 × 10⁷, 2·0 × 10⁷ and 3·0 × 10⁷ conidia ml^?1, respectively. Metarhizium anisopliae isolate ICIPE 69 produced significantly more conidia than M. anisopliae isolates ICIPE 7 and ICIPE 20. Internally transcribed spacers sequences alignment showed differences in nucleotides composition, which can partly explain differences in virulence. Conclusion: These results coupled with the previous ones on virulence and field efficacy against other species of thrips make M. anisopliae isolate ICIPE 69 a good candidate. Significance and Impact of the Study: Metarhizium anisopliae isolate ICIPE 69 can be suggested for development as fungus‐based biopesticide for thrips management.     

Isolation of entomopathogenic fungi from Northern Thailand and their production in cereal grains

Spore productivity in six entomopathogenic fungal strains isolated from insect cadavers at four locations in Chiang Mai province was evaluated in five cereal grains: white-rice, wheat, rye, corn and sorghum. According to sequence analysis of the internal transcribed spacer regions of these isolates, they were closely related to Beauveria bassiana (2 isolates), Metarhizium flavoviride (1 isolate), Metarhizium anisopliae (1 isolate), Paecilomyces lilacinus (1 isolate) and Isaria tenuipes (1 isolate). Among all fungal isolates, the maximum amount of spores (530.0?×?10⁹ conidia/g) was yielded P. lilacinus CMUCDMT02 on sorghum grain followed by white-rice (399.3?×?10⁹ conidia/g). Moreover, the highest number of spore in M. flavoviride was 102.8?×?10⁹ conidia/g sorghum whereas white-rice yielded the greatest amount of spore for B. bassiana CMUCDMF03 (141.0?×?10⁹ conidia/g) after 60?days incubation. The fungal growth rate was found highest in corn for all strains and rye showed the lowest with the exception of P. lilacinus CMUCDMT02 among the tested grains. Spore viability was over 80?% for all isolates that had been inoculated for 60?days. Fungal conidia suspension of P. lilacinus obtained highest virulence against Bactrocera spp. at a concentration of 1?×?10⁶ spore/ml. The strains isolated, exhibited good production of conidia suggesting a promising strategy for the mass production of inoculum as biocontrol agents with low production cost.     

Production of inulinase by solid-state fermentation: effect of process parameters on production and preliminary characterization of enzyme preparations

This work was aimed at producing inulinase by solid-state fermentation of sugarcane bagasse, using factorial design to identify the effect of corn steep liquor (CSL) and soybean bran concentration, particle size of bagasse and size of inoculum. Maximum inulinase activity achieved was 250 U per g of dry substrate (gds) at 20% (w/w) of CSL, 5% (w/w) of soybean bran, 1 × 10¹⁰ cells mL⁻¹ and particle size of bagasse in the range 9/32 mesh. The use of soybean bran decreased the time to reach maximum activity from 96 to 24 h and the maximum productivity achieved was 8.87 U gds⁻¹ h⁻¹. The maximum activity was obtained at pH 5.0 and 55.0°C. Within the investigated range, the enzyme extract was more thermostable at 50.0°C, showing a D-value of 123.1 h and deactivation energy of 343.9 kJ gmol⁻¹. The extract showed highest stability from pH 4.5 to 4.8. Apparent K _m and V _max are 7.1 mM and 17.79 M min⁻¹, respectively.     

Effect of entomopathogenic fungus <Emphasis Type="Italic">Metarhizium robertsii</Emphasis> on non-target organisms,water bugs (Heteroptera: Corixidae,Naucoridae, Notonectidae)

The influence of the fungus Metarhizium robertsii Bischoff, Rehner and Humber on the mortality of four water bug species, Cymatia coleoptrata (Fabricius), Sigara assimilis (Fieber), Ilyocoris cimicoides cimicoides (Linnaeus), and Notonecta reuteri Hungerford, and bloodsucking mosquito Anopheles messeae Falleroni, was investigated under various concentrations of conidia and different treatment types. We found that the mortality of adults of the water bug species was similar or higher than that of A. messeae, with C. coleoptrata and S. assimilis being more susceptible to M. robertsii than N. reuteri, I. c. cimicoides, and the mosquito A. messeae. Treatment with dry conidia at concentrations of 5 × 10⁴ and 5 × 10⁵ conidia/ml caused higher mortality of the water bug species than did treatment at the same concentrations with conidia in an aqueous suspension. In contrast, higher concentrations (5 × 10⁶ conidia/ml) led to higher mortality after treatment with the aqueous suspension, relative to treatment with dry conidia. Our studies showed that water bugs exhibited the classical development of a mycosis with hemocoel colonization, mummification, and conidia formation on cadavers directly on the surface of the water. Possible changes in invertebrate communities in aquatic ecosystems after treatment with Metarhizium are discussed.     

Studies on the production of nigerloxin using agro-industrial residues by solid-state fermentation

Nigerloxin, a new and potent lipoxygenase inhibitor, was discovered in our laboratory through solid-state fermentation of wheat bran by Aspergillus niger V. Teigh (MTCC-5166). The aim of this study is to investigate the possibility of using different agro-industrial residues as nutritional supplements along with wheat bran to enhance the production of nigerloxin. Nigerloxin produced by SSF was quantified spectrophotometrically at 292 nm. The results indicate that the inhibitor production was influenced by the type of solid substrate supplemented, moisture content, pH and size of the inoculum. Individually optimized supplements were tested in different combinations to determine their effects on nigerloxin production. A twofold increase in the production of nigerloxin (4.9 ± 0.3 mg gds⁻¹) was achieved by supplementing wheat bran with 10% w/w sweet lemon peel and 5% v/w methanol at optimized process parameters, that is, an initial moisture content of 65% v/w and incubation period of 6 days with an initial inoculum size of 2 ml (8 × 10⁵ spores gds⁻¹). Nigerloxin production was stable between pH of 4 and 5.     

Mixture design as first step for improved glutaminase production in solid-state fermentation by isolated Bacillus

Aim: Investigation of mixture‐design impact on glutaminase production by isolated Bacillus sp. Methods and Results: An augmented simplex centroid design was used to optimize a three (wheat bran, Bengal gram husk and palm seed fibre) component mixture for glutaminase production. Selected substrate materials showed impact on glutaminase production values at individual level by Bengal gram husk [2789 U gds^?1 (gram dry substrate] and in two‐level combination with wheat bran and Bengal gram husk (maximum of 3300 U gds^?1). Conclusion: Bengal gram husk is the most suitable substrate medium for glutaminase production by Bacillus sp. Maximum glutaminase production is achieved using solid‐substrate mixture at two‐level combinations in the ratio of 66 : 34 for Bengal gram husk and wheat bran, respectively. Significance and Impact of the Study: The present study has significance in large‐scale production of glutaminase at commercial level with the use of multi‐substrate rather than single‐substrate/support material.     

Isolation and pathogenicity of some South African entomopathogenic fungi (Ascomycota) against eggs and larvae of Cydia pomonella (Lepidoptera: Tortricidae)

Virulent entomopathogenic fungal strains against Cydia pomonella (Lepidoptera: Tortricidae) were isolated and identified in the Western Cape Province of South Africa. Thirty-nine isolates belonging to six species were obtained using the insect bait method. Generally, Metarhizium robertsii (Ascomycota: Hypocreales) was the most frequently encountered species representing 51% of the total number of isolates collected from the soil samples. This is the first report of M. robertsii from southern Africa. Mortality data from an immersion bioassay indicated that the 39 fungal isolates were pathogenic against fifth instar larvae of C. pomonella inducing 47–85% insect mortalities. Two M. robertsii strains, MTL151 and GW461, induced 85% larval mortality and were selected for further evaluations. The exposure of freshly laid eggs to wax papers that were pre-treated with fungal spores ranging from 10³ to 10⁸ spores ml^?1 of MTL151 and GW461 resulted in a significant reduction of egg hatchabilities, from 95 to 66% and 93 to 71%, respectively as spore concentration increased. First instar larval neonates were exposed to apples that were pre-sprayed topically with varied conidia suspensions (10³?10⁸ spores ml^?1). The mean percentage of participating apples with larvae in their cores/flesh significantly reduced from 53 to 10% (MTL151) and 76 to 10% (GW461) of 10 apples, and a concurrent decrease in the incidence of apple fruit rot was observed as conidia concentration increased. Up to 90% of apples treated with 1 × 10⁸ spores ml^?1 had no larvae present in their cores and this result compared favourably with the commercial pesticide Fruitfly®, containing the active ingredient cypermethrin (20 g/l) used at a recommended dose of 0.25 ml/250 ml of water.     

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.     

Evaluation of inert and organic carriers for <Emphasis Type="Italic">Verticillium lecanii</Emphasis> spore production in solid-state fermentation

Growth and sporulation of Verticillium lecanii on inert and organic carriers (sugar-cane bagasse, corncob, rice straw, polyurethane foam and activated carbon) in a solid-state fermentation process was studied. Sugar-cane bagasse and polyurethane foam produced 10¹⁰ spores g⁻¹ dry carrier whereas corncob, rice straw, and activated carbon yielded, respectively 8 × 10⁹, 4 × 10⁹, and 3 × 10⁸ spores g⁻¹. Chitinase activity of the conidia was in the following order: sugar-cane bagasse (3.3 U mg⁻¹) > wheat bran (3.0 U mg⁻¹) > polyurethane foam (2.7 U mg⁻¹). There was no significant difference (2.5–2.7 U mg⁻¹) in the proteinase activity among the conidia from the three cultures. Scanning electron microscopy shows that aerial mycelium freely penetrated into the internal area of polyurethane foam. Sugar-cane bagasse provided enough area for vegetative hyphae to attach. Of the carriers analyzed, polyurethane foams and sugar-cane bagasse were the best carriers for V. lecanii growth and spore production.     

Ethanol production from alkali-treated rice straw via simultaneous saccharification and fermentation using newly isolated thermotolerant <Emphasis Type="Italic">Pichia kudriavzevii</Emphasis> HOP-1

In this study, simultaneous saccharification and fermentation (SSF) was employed to produce ethanol from 1% sodium hydroxide-treated rice straw in a thermostatically controlled glass reactor using 20 FPU gds⁻¹ cellulase, 50 IU gds⁻¹ β-glucosidase, 15 IU gds⁻¹ pectinase and a newly isolated thermotolerant Pichia kudriavzevii HOP-1 strain. Scanning electron micrograph images showed that the size of the P. kudriavzevii cells ranged from 2.48 to 6.93 μm in diameter while the shape of the cells varied from oval, ellipsoidal to elongate. Pichia kudriavzevii cells showed extensive pseudohyphae formation after 5 days of growth and could assimilate sugars like glucose, sucrose, galactose, fructose, and mannose but the cells could not assimilate xylose, arabinose, cellobiose, raffinose, or trehalose. In addition, the yeast cells could tolerate up to 40% glucose and 5% NaCl concentrations but their growth was inhibited at 1% acetic acid and 0.01% cyclohexamide concentrations. Pichia kudriavzevii produced about 35 and 200% more ethanol than the conventional Saccharomyces cerevisiae cells at 40 and 45°C, respectively. About 94% glucan in alkali-treated rice straw was converted to glucose through enzymatic hydrolysis within 36 h. Ethanol concentration of 24.25 g l⁻¹ corresponding to 82% theoretical yield on glucan basis and ethanol productivity of 1.10 g l⁻¹ h⁻¹ achieved using P. kudriavzevii during SSF hold promise for scale-up studies. An insignificant amount of glycerol and no xylitol was produced during SSF. To the best of our knowledge, this is the first study reporting ethanol production from any lignocellulosic biomass using P. kudriavzevii.     

Entomopathogenic fungus as a biological control agent against Rhyzopertha dominica F. (Coleoptera: Bostrychidae) on stored wheat

To evaluate the pathogenicity of Metarhizium anisopliae (Metschinkoff) Sorokin (Deuteromycotina: Hyphomycetes) a bioassay was designed under laboratory conditions against Rhyzopertha dominica F. (Coleoptera: Bostrychidae) on stored wheat. The fungus was applied at the dose rates of 8 × 10³, 8 × 10⁵, 8 × 10⁷ and 8 × 10⁹ conidia/kg of wheat and the bioassay was conducted at 25°C with 60% relative humidity. The data regarding the mortality was recorded after 7 and 14 days exposure intervals. All the treatments gave the significant mortality of R. dominica and M. anisopliae of 8 × 10⁹ conidia/kg was found to be the most effective after a 14-day exposure interval. There was greater production of progeny when the low rate of M. anisopliae was applied to wheat. Overall, our study showed that M. anisopliae is vigorous when applied at a high dose rate which revealed an effective control of R. dominica and also played a pivotal role in the integrated pest management program (IPM) of stored wheat insect pests.     

Cyclic pollen production in Cedrus deodara

Based on a seven-year study of pollen production and release in two different natural populations of Cedrus deodara from Garhwal Himalaya, India, we determined that pollen output follows a two-year cycle. Pollen productivity determinations considered various sources of variability, including the number of pollen strobili per branch, strobili per tree, microsporophylls per tree and pollen grains per tree. Each of these parameters revealed significant year-to-year and population effects. Microsporangium dehiscence took from 2.5 to 3.5 days. Maximum dehiscence was observed between 12:00 and 14:00 h, which coincides with diurnal highest temperature and lowest relative humidity. Annual production of pollen per tree varied from averages of 4.7 × 10⁹, 7.2 × 10⁹ and 5.1 × 10⁹ in years of low production, with alternate high years, producing 12.6 × 10⁹, 14.1 × 10⁹, 13.3 × 10⁹ and 14.0 × 10⁹ pollen grains per tree. Annual pollen production in individual trees of C. deodara ranged from 1.4 × 10⁹ to 22.3 × 10⁹.