Potential of a Dried Rice/Mycelium Formulation of Entomopathogenic Fungi to Suppress Subterranean Pests in Small Fruits

The potential of a dried rice/mycelium formulation of three species of entomopathogenic fungi ( Metarhizium anisopliae, Beauveria bassiana and Paecilomyces farinosus ) was assessed according to five criteria: storage life, field efficacy against two subterranean pests of cranberry, persistence and efficacy against first-instar black vine weevil larvae in potted strawberry and field persistence in a cranberry bog. The number of conidia sporulated from the formulation did not decline between 4 and 20 days. In small plot field trials, the numbers of black vine weevil, Otiorhynchus sulcatus (F.), were significantly lower in plots treated with 1000 gm - 2 of formulated M. anisopliae than in untreated plots. In similar trials, the numbers of adult cranberry girdler, Chrysoteuchia topiaria (Zeller), inside emergence cages were significantly lower on plots treated with the formulated M. anisopliae than on untreated plots. The formulation suppressed first-instar black vine weevil larvae in potted strawberry regardless of the species of entomopathogenic fungus or the post-inoculation interval. M. anisopliae was periodically isolated for up to 637 days from plots in cranberry bogs treated with the formulation. In general, the dried rice/mycelium formulation may be a useful fungal mycopesticide for the management of subterranean pests of small fruits, especially if applied at high rates to areas of serious pest infestation.     

Production process reproducibility and product quality consistency of transient gene expression in HEK293 cells with anti-PD1 antibody as the model protein

Demonstration of reproducibility and consistency of process and product quality is one of the most crucial issues in using transient gene expression (TGE) technology for biopharmaceutical development. In this study, we challenged the production consistency of TGE by expressing nine batches of recombinant IgG antibody in human embryonic kidney 293 cells to evaluate reproducibility including viable cell density, viability, apoptotic status, and antibody yield in cell culture supernatant. Product quality including isoelectric point, binding affinity, secondary structure, and thermal stability was assessed as well. In addition, major glycan forms of antibody from different batches of production were compared to demonstrate glycosylation consistency. Glycan compositions of the antibody harvested at different time periods were also measured to illustrate N-glycan distribution over the culture time. From the results, it has been demonstrated that different TGE batches are reproducible from lot to lot in overall cell growth, product yield, and product qualities including isoelectric point, binding affinity, secondary structure, and thermal stability. Furthermore, major N-glycan compositions are consistent among different TGE batches and conserved during cell culture time.

     

Fermentation and microencapsulation of the nematophagous fungus <Emphasis Type="Italic">Hirsutella rhossiliensis</Emphasis> in a novel type of hollow beads

In this work, fermentation and formulation aspects of the nematophagous fungus Hirsutella rhossiliensis BBA were investigated. When incubated in 2% (w/w) glucose and 0.5% (w/w) yeast extract medium in a 1-L Erlenmeyer flask without baffles, heavy pellet formation was observed. Only 40% of the mycelium had a size less than 500 μm. When a flask with three baffles was used, the portion of mycelium <500 μm rose to 95%. In the next step, the influence of aeration rate and stirrer speed on production of finely dispersed mycelium in a stirred tank reactor was investigated. The best fermentation results were obtained at 0.4 vvm and 400 rpm stirrer speed with 90% mycelium <500 μm and 5 g/L biomass. Then, mycelium was microencapsulated in hollow beads based on sulfoethylcellulose (SEC). Experiments on the capsule nutrient reservoir showed that 15% (w/w) corn gluten and 0.5% (w/w) yeast extract could be replaced with 3% (w/w) autoclaved baker's yeast which was never used as capsule additive before. Radial growth of mycelium out of dried hollow beads containing 1% (w/w) biomass and 3% (w/w) baker's yeast was faster than for alginate beads containing equivalent amounts of biomass and yeast indicating a higher bio-control potential.     

Validation of a 30-year-old process for the manufacture of l-asparaginase from Erwinia chrysanthemi

A 30-year-old manufacturing process for the biologic product l-asparaginase from the plant pathogen Erwinia chrysanthemi was rigorously qualified and validated, with a high level of agreement between validation data and the 6-year process database. l-Asparaginase exists in its native state as a tetrameric protein and is used as a chemotherapeutic agent in the treatment regimen for Acute Lymphoblastic Leukaemia (ALL). The manufacturing process involves fermentation of the production organism, extraction and purification of the l-asparaginase to make drug substance (DS), and finally formulation and lyophilisation to generate drug product (DP). The extensive manufacturing experience with the product was used to establish ranges for all process parameters and product quality attributes. The product and in-process intermediates were rigorously characterised, and new assays, such as size-exclusion and reversed-phase UPLC, were developed, validated, and used to analyse several pre-validation batches. Finally, three prospective process validation batches were manufactured and product quality data generated using both the existing and the new analytical methods. These data demonstrated the process to be robust, highly reproducible and consistent, and the validation was successful, contributing to the granting of an FDA product license in November, 2011.     

两株椰心叶甲分离物的鉴定及其系统发育分析

采用形态学方法对2株从自然罹病死亡的椰心叶甲虫尸上分离到的致病菌株Dz01和Ma4进行了鉴定,发现2个菌株在菌丝、瓶梗和分生孢子等形态特征上与金龟子绿僵菌小孢变种基本一致,可将2个菌株鉴定为金龟子绿僵菌小孢变种。基于Dz01和Ma4菌株和其它31个代表绿僵菌主要种或变种菌株rDNA上ITS1-5.8S-ITS2区序列构建的最大简约树显示,Dz01和Ma4菌株均聚在金龟子绿僵菌小孢变种所构成的分支中,这为2个菌株形态学鉴定结果提供了分子依据。     

Propagules of arbuscular mycorrhizal fungi in a secondary dry forest of Oaxaca, Mexico

Plant cover loss due to changes in land use promotes a decrease in spore diversity of arbuscular mycorrhizal fungi (AMF), viable mycelium and, therefore, in AMF colonization, this has an influence in community diversity and, as a consequence, in its recovery. To evaluate different AMF propagules, nine plots in a tropical dry forest with secondary vegetation were selected: 0, 1, 7, 10, 14, 18, 22, 25, and 27 years after abandonment in Nizanda, Oaxaca, Mexico. The secondary vegetation with different stages of development is a consequence of slash and burn agriculture, and posterior abandonment. Soil samples (six per plot) were collected and percentage of AMF field colonization, extrarradical mycelium, viable spore density, infectivity and most probable number (MPN) ofAMF propagules were quantified through a bioassay. Means for field colonization ranged between 40% and 70%, mean of total mycelium length was 15.7 +/- 1.88 mg(-1) dry soil, with significant differences between plots; however, more than 40% of extracted mycelium was not viable, between 60 and 456 spores in 100 g of dry soil were recorded, but more than 64% showed some kind of damage. Infectivity values fluctuated between 20% and 50%, while MPN showed a mean value of 85.42 +/- 44.17 propagules (100 g dry soil). We conclude that secondary communities generated by elimination of vegetation with agricultural purposes in a dry forest in Nizanda do not show elimination of propagules, probably as a consequence of the low input agriculture practices in this area, which may encourage natural regeneration.     

Field Trial with the Entomopathogenic Fungus Metarhizium anisopliae var. acridum against Bands of the Grasshopper Rhammatocerus schistocercoides in Brazil

The efficacy of a mycoinsecticide formulated in vegetable oil was tested in Brazil against the grasshopper Rhammatocerus schistocercoides . A set of experiments was conducted in the Chapada dos Parecis region (Mato Grosso state), a permanent zone of outbreaks for this pest. Experiments were performed in zones of natural vegetation, against grasshopper bands in the third nymphal instar. Three nymphal bands were treated with a mycoinsecticide formulation based on conidia of the entomopathogenic fungus Metarhizium anisopliae var. acridum ( =M. flavoviride ), strain CG 423. Three non-treated bands were used as control. The application was made with the aid of a hand-held ULV sprayer adjusted to deliver 2 l of the formulation ha -1 , each containing 1 ×10 13 conidia. Treatments were limited to the surface of the grasshopper bands and their immediate borders (5-10 m). The efficacy of the mycoinsecticide was evaluated through band survival after treatment (grasshopper numbers, surface, density, behaviour and daily movement of the band), allowing the insects to move freely in their natural environment. Insects were regularly surveyed and maintained in the laboratory, allowing estimates of the infection rate. Field and laboratory studies showed a clear effect of the product 10 days after treatment. At 14 days post-spraying, mortality caused by the mycoinsecticide in the field was approximately 88%.     

Stabilization of a recombinant human epidermal growth factor parenteral formulation through freeze-drying

Development studies were performed to design a pharmaceutical composition that allows the stabilization of a parenteral rhEGF formulation in a lyophilized dosage form. Unannealed and annealed drying protocols were tested for excipients screening. Freeze-dry microscopy was used as criterion for excipients and formulation selection; as well as to define freeze-drying parameters. Excipients screening were evaluated through their effect on freeze-drying recovery and dried product stability at 50 °C by using a comprehensive set of analytical techniques assessing the chemical stability, protein conformation and bioactivity. The highest stability of rhEGF during freeze-drying was achieved by the addition of sucrose or trehalose. After storing the dried product at 50 °C, the highest stability was achieved by the addition of dextran, sucrose, trehalose or raffinose. The selected formulation mixture of sucrose and dextran could prevent protein degradation during the freeze-drying and delivery processes. The degradation rate assessed by RP-HPLC could decrease 100 times at 37 °C and 70 times at 50 °C in dried with respect to aqueous formulation. These results indicate that the freeze-dried formulation represents an appropriate technical solution for stabilizing rhEGF.     

Near‐infrared spectroscopic evaluation of lyophilized viral vaccine formulations

This article examines the applicability of near‐infrared spectroscopy (NIRS) to evaluate the virus state in a freeze‐dried live, attenuated vaccine formulation. Therefore, this formulation was freeze‐dried using different virus volumes and after applying different pre‐freeze‐drying virus treatments (resulting in different virus states): (i) as used in the commercial formulation; (ii) without antigen (placebo); (iii) concentrated via a centrifugal filter device; and (iv) stressed by 96 h exposure to room temperature. Each freeze‐dried product was measured directly after freeze‐drying with NIR spectroscopy and the spectra were analyzed using principal component analysis (PCA). Herewith, two NIR spectral regions were evaluated: (i) the 7300–4000 cm^?1 region containing the amide A/II band which might reflect information on the coated proteins of freeze‐dried live, attenuated viruses; and (ii) the C–H vibration overtone regions (10,000–7500 and 6340–5500 cm^?1) which might supply information on the lipid layer surrounding the freeze‐dried live, attenuated viruses. The different pre‐freeze‐drying treated live, attenuated virus formulations (different virus states and virus volumes) resulted in different clusters in the scores plots resulting from the PCA of the collected NIR spectra. Secondly, partial least squares discriminant analysis models (PLS‐DA) were developed and evaluated, allowing classification of the freeze‐dried formulations according to virus pretreatment. The results of this study suggest the applicability of NIR spectroscopy for evaluating live, attenuated vaccine formulations with respect to their virus pretreatment and virus volume. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1573–1586, 2013     

Method of production of the dried highly dispersed vaccine of Newcastle disease virus

Experimental batches of dried dust-like vaccine on the basis of Newcastle disease virus, strain La Sota, have been prepared. The technology of the preparation of the vaccine includes the lyophilization of the technical fluid of the culture under study, milling dried biomass and mixing the pulverized biomass with excipient. As revealed in the process of this work, lyophilization ensures high concentration of the virus in dried biomass and its pulverization, a high content of the target fraction of biomass particles in the vaccine preparation with the moderate inactivation of the bioagent. By mixing the pulverized biomass with excipient the required concentration of the viable virus in the finished product may be achieved. The viability of the bioagent during the storage of the dried vaccine under conditions, determined by optimum storage temperature and time, has been found satisfactory.     

Formation of glyceryl 2-mononitrate by regioselective bioconversion of glyceryl trinitrate: efficiency of the filamentous fungus Phanerochaete chrysosporium

Various microorganisms have been evaluated for their ability to hydrolyze glyceryl trinitrate (GTN) to glyceryl dinitrates and mononitrates. Provided that the GTN extracellular concentration was under the lethal dose, metabolite formation and regioselectivity depend on the nature of the strain used. In particular, Phanerochaete chrysosporium at a sublethal dose (3 mM) converts GTN into 1,2-glyceryl dinitrate and 2-glyceryl mononitrate (2-GMN) with a 80% regioselectivity in both steps. This bioconversion, when carried out in fermentors at 28 degrees C, allowed formation of 2-GMN at a rate of 12 mumol/h/g of dried mycelium. Successive batches of 3 mM GTN could be converted into 2-GMN as long as consecutive additions of glycerol or glucose were effected to ensure cell survival and the efficiency of the enzymatic system involved.     

Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum

AIMS: To examine the ability of Agrobacterium to attach to Metarhizium anisopliae var. acridum strain CG423 under co-cultivation and to develop an Agrobacterium-mediated method of gene delivery into strain CG423, a promising agent for biological control of grasshoppers. METHODS AND RESULTS: The co-cultivation of Agrobacterium tumefaciens and M. anisopliae var. acridum was analysed under scanning electron microscopy. We observed that Agrobacterium attached to and formed aggregates around Metarhizium conidia and germ tubes. We also observed the occurrence of fibril-like structures connecting neighbouring bacterial-fungal cells. The Agrobacterium-mediated transformation was applied using two binary vectors carrying a benomyl resistance gene as a selection marker. The efficiency of transformation was up to 53 transformants per 10(5) target conidia. High mitotic stability of the transformants (89-97%) was demonstrated after five successive transfers on non-selective media. Molecular analysis revealed the occurrence of high frequency of gene conversion. CONCLUSIONS: In our study, we report that A. tumefaciens strain AGL-1 attaches to and genetically transforms the entomopathogenic fungus Metarhizium anisopliae var. acridum. SIGNIFICANCE AND IMPACT OF THE STUDY: We report for the first time, the attachment of Agrobacterium to fungal cells opening new avenues for the study of this essential step of the T-DNA transfer process. Considering the efficiency of the transformation protocol herein described, this is a useful tool for gene disruption in M. anisopliae var. acridum.     

Biological control of black vine weevil Otiorhynchus sulcatus in Ireland using Heterorhabditis megidis

Heterorhabditis megidis (UK211) was applied against black vine weevil (BVW, Otiorhynchus sulcatus) in potted plants in a polyethylene (2002) or glasshouse (2003) and in field planted strawberries (2003). Both potted and field strawberries were artificially infested with BVW larvae. In a 2002 pot planting in the polyethylene house, a single drench application of 25,000 H. megidis infective juveniles per plant in 50 ml of water in mid September, reduced the number of BVW larvae to 1.8/20 plants. A second application in early October gave a reduction of 0.2/20 plants and in the third application, the following March no live weevils were recovered, compared to the control which had 8.2 larvae/20 plants. In a 2003 pot planting in a glasshouse, similar treatments gave a reduction of 5.2, 5.4 and 0.8 larvae/20 plants, respectively, compared to the control where 26.2 larvae/20 plants were recovered. In an artificially, BVW infested field trial, similar treatments gave a reduction to 2.2 larvae/20 plants in the single September treatment, and 2 larvae/20 plants in the single October treatment. The double (September and October) application reduced BVW larvae further to 1.6/20 plants and the triple (September, October and April) application to 0.4 larvae/20 plants, compared to the control where four larvae corresponded to every 20 plants. There was, therefore, little difference between the single and double autumn treatments indoors or in the field, and it mattered little whether the single application in the field was made in September or October under the conditions of 2003. Early spring application gave a significant reduction in BVW in each of the three experiments.     

Comparison of an Organophosphate Insecticide with a Mycoinsecticide for the Control of Oedaleus senegalensis (Orthoptera: Acrididae) and Other Sahelian Grasshoppers at an Operational Scale

Operational scale field trials were conducted in 1996 and 1997, in the east of the Niger Republic, on 50 and 800 hectare plots, to compare the efficacy of an oil based formulation of the entomopathogenic fungus, Metarhizium anisopliae (flavoviride) var. acridum (Deuteromycotina: Hyphomycetes) with fenitrothion for the control of Sahelian grasshoppers. The Senegalese Grasshopper Oedaleus senegalensis Krauss was the most abundant species in the trials. M. anisopliae was applied at 5 x 1012 spores ha-1 at volume application rates of 2 and 0.5 l ha-1 in successive years. Fenitrothion was applied at 220 g/ha-1 at 1.25 and 0.22 l ha-1 volume application rates. Ultra low volume equipment mounted on a vehicle (1996) or a fixed wing aircraft (1997) was used for application. The M. anisopliae treatment reduced the grasshopper population significantly after 7 days and by 93% within 16 days. Fenitrothion caused a population reduction of more than 90% shortly after application, but due to immigration, the grasshopper population recovered to the initial level within 16 days. Grasshoppers treated with the fungus and given the opportunity to thermoregulate in the sun died more slowly than grasshoppers incubated in the shade. The survival of spores in the spray residue of the M. anisopliae plots assessed by exposing grasshoppers to the sprayed vegetation at intervals and monitoring disease levels during subsequent laboratory incubation, showed the spray residue to remain highly infective, for three weeks after spraying. At the end of the 1997 season, egg pod density and viability in the plot treated with the fungus was reduced compared with both untreated and the fenitrothion plots. Compared with the existing practice of large-scale treatment of grasshopper infestations with fenitrothion, use of M. anisopliae would not only be safer to mammals and less damaging to non-target organisms, but also be more effective in the long-term control of grasshoppers.     

Long-term Field Efficacy of the Entomogenous Fungus Metarhizium anisopliae against the Subterranean Scarab, Adoryphorus couloni

The efficacy of Metarhizium anisopliae DAT F-001 against Adoryphorus couloni was examined over 4 years on a sheep grazing property ('Inverell') in central Tasmania. Three generations of the primary A. couloni population and two generations of the overlapping population were studied. Application of 5.1 0.7 104 M. anisopliae spores g-1 of soil (2 cm below the soil surface), during midwinter 1989, when the primary population was in the middle of the L3 larval stage, resulted in 30.3% fewer larvae in treated plots by 21 weeks and 57.8% less pupae by 27 weeks. The population decline was consistent with a mortality model developed from laboratory data. In the two subsequent generations of the primary population, there were 63.2% (1991) and 45.0% (1993) fewer larvae in the treated plots before the damaging L3 stage. The two sequential generations of the overlapping A. couloni larval populations had 68% (1990) and 65% (1992) fewer larvae in the treated plots than in the untreated plots. Reductions in larval numbers led to a greater retention of sown perennial grasses, reduced weed invasion and a 23% increase in pasture productivity in the autumn of 1992. Incorporation of M. anisopliae into the soil did not reduce the numbers of non-target invertebrates. The level of M. anisopliae DAT F-001 in the pasture remained at levels close to the applied concentration (5.1 0.7 colony-forming units g-1 of soil), but increased 10-fold when mummified L3 larval, prepupal and pupal cadavers were present from January to June 1990. The level of the fungus in the soil was still twice the original applied concentration at the conclusion of sampling in March 1992.     

Effect of formulated Metarhizium anisopliae on eggs and eclosing nymphs of Triatoma infestans

Little is known about the ovicidal effects of fungi that attack nymphs and adults of triatomine vectors. A combined formulation of Metarhizium anisopliae IP 46 conidia prepared with diatomaceous earth (DE) and vegetable oil was tested against eggs of Triatoma infestans. Eggs were highly susceptible to fungal infection at relative humidity close to saturation [>98% relative humidity (RH)] but not at 75% RH regardless of the formulation applied. Susceptibility of eggs decreased with longer post‐ovipositional embryonation periods before treatments. The eventual eclosion of nymphs was best suppressed by application of conidia prepared with DE + oil and at a >98% RH incubation. Moreover, nymphs were less affected by the fungus when exposed for only a 24‐h period after eclosion to a treated surface than individuals that were in constant contact with the conidia. These findings contribute to a better understanding of the potential of M. anisopliae as an agent against all developmental stages of T. infestans.     

Influence of the application methods and doses on the susceptibility of black vine weevil larvae Otiorhynchus sulcatus to Metarhizium anisopliae in field-grown strawberries

Entomopathogenic fungi are commercially available for the control of insect pests, including the black vine weevil (BVW) Otiorhynchus sulcatus Fabricius (Coleoptera:Curculionidae). However, Metarhizium anisopliae (Metsch.) Sorokin (Clavicipitaceae) has not been used to control BVW in field-grown strawberries. Field trials were conducted in different locations in the UK during 2009–2010 to evaluate the different formulations (granular vs. drench) and application methods (premixed, drench, bare root treatment) of commercial strain of M. anisopliae Met52^® (=F52), the entomopathogenic nematodes and the organophosphate insecticide Cyren^® against BVW. The highest dose (10¹⁴ cfu ha^?1) tested provided significantly better control than the intermediate (10¹³ cfu ha^?1) or low (10¹² cfu ha^?1) doses. BVW larval control at the high, intermediate and low doses was 71–96, 40–75 and 6–11 %, respectively. Premixing, drench or bare root treatment with Met52^® gave similar levels of BVW control. Irrespective of the application methods or soil types, the high dose rate of Met52^® provided the best control. Significantly high larval control was achieved (78–97 %) when chlorpyrifos was applied at planting than eight weeks post planting (53 %). There were significant differences in BVW control between Met52^® (88 %) and reduced doses of Heterorhabditis bacteriophora Poinar (20–29 %) or Steinernema kraussei Steiner (39–75 %) when applied alone. However, when used together, low dose of S. kraussei and Met52^® provided 100 % control of BVW larvae. This study shows that Met52^® has considerable potential for the control of BVW larvae in commercial field-grown strawberry, thereby offering an environmentally benign alternative to chemical insecticides.     

Preparation of a high-immunoglobulin product from bovine colostrum

An effective colostrum management programme is the most important factor in determining the health and survival of the neonatal calf. Commercially available colostrum replacers (CR) and colostrum supplements (CS) are an alternative to colostrum on farms that do not have an adequate, high-quality colostrum supply, or those farms that want to prevent transmission of disease from cow to calf. The present study aimed to obtain a high immunoglobulin (Ig), dried bovine colostrum product that could be used as a CR or CS for dairy calves. Dried whey was made from 6 batches of colostrum and an additional 6 batches were used to produce dried whey and curds. Dried whey had higher IgG concentration (P<0.05) and lactose (P<0.05), and less fat (P<0.05) compared to curds or the original colostrum. There was a strong linear relationship between initial colostrum IgG concentration and whey (R² = 0.86) with approximately 0.45 of the initial colostral IgG residing in curd. The high IgG and the composition of colostrum whey powder suggests it could be an effective CS product for use with dairy calves. The high fat and IgG content of the curd by-product indicate that it might be a potential weaning supplement in piglets or even a product for human consumption.     

The entomopathogenic nematode Steinernema kraussei and Metarhizium anisopliae work synergistically in controlling overwintering larvae of the black vine weevil,Otiorhynchus sulcatus,in strawberry growbags

Previously, the combination of reduced rate of entomopathogenic nematodes (EPN) and fungus caused additive or synergistic mortality to third-instar black vine weevil (BVW), Otiorhynchus sulcatus. In this study, we examined this interaction in unheated glasshouses during winter and compared a combination of commercial formulation of a cold-tolerant EPN, S. kraussei (Nemasys L?) and fungus Metarhizium anisopliae strain V275 against overwintering third-instar BVW. The combination of M. anisopliae with S. kraussei at a rate of 1×10¹⁰ conidia+250,000 nematodes/growbag resulted in additive or synergistic effects, providing 100% control of overwintering larvae.     

Production ofBeauveria bassiana [Deuteromycotina: Hyphomycetes] in different liquid media and subsequent conidiation of dry mycelium

Mycelium ofBeauveria bassiana can be grown in liquid culture, filtered, and the mycelium dried. After rehydration the mycelium sporulates. Two carbohydrate sources (sucrose and maltose), and one nitrogen/vitamin source (yeast extract) were tested for mycelium growth and subsequent conidial production. Maximum mycelium growth (12.31 mg/ml), in liquid culture, was in the sucrose (3.5%)/yeast extract (3.5%) medium, but mycelium from a maltose (2%)/yeast extract (0.75%) medium produced the maximum of 4.62×10⁶ conidia/mg dry mycelium after incubation in moist Petri dishes. Using the data on mycelium yield (in liquid culture) and conidial production (by dry mycelium) it is calculated that the sucrose (3.5%)/yeast extract (3.5%) and the maltose (2%)/yeast extract (0.75%) media produce most conidia per media volume (an equivalent of 3.52–3.72×10⁷ conidia/ml).