Conidia Of Entomopathogenic Fungi Research Articles

The Combination of Enzymes and Conidia of Entomopathogenic Fungi against Aphis gossypii Nymphs and Spodoptera frugiperda Larvae.

The slow action of fungi is one of the biggest challenges in using entomopathogenic fungi. A promising alternative to reduce the time of action is to combine conidia with extracellular enzymes. This study aimed to characterize the production of Pr1 subtilisin protease and lipases by Beauveria bassiana and Metarhizium anisopliae in different culture media and to evaluate the efficiency of the enzymatic treatment against Aphis gossypii and Spodoptera frugiperda. The isolates were cultivated in five different liquid cultures, and, after 7 days, the culture was filtered and centrifuged, and the activity of the Pr1 and lipases was measured. The fungi cultured in a Luria-Bertani broth medium had the highest activity of proteases and lipases. The mortality of A. gossypii nymphs treated with conidia 7 days after the treatment was 39% (JEF-410), 76.5% (JEF-492), 74.8% (ERL-836), and 70.9% (JEF-214). The B. bassiana JEF-410 supernatant combined with conidia increased the fungal virulence at day 5 and day 6 after treatment. When S. frugiperda larvae were treated with B. bassiana JEF-492 conidia combined with its supernatant, the time of infection was shorter compared to the larvae treated with conidia only. Once the supernatant was incubated at 37 °C, the relative activity decreased from 100% to 80% after 2 h and to 45% after 24 h. The results suggest that the supernatant of entomopathogenic fungi may be formulated and used as a biopesticide in an efficient strategy for the biological control of pests.

The Dissemination of Metarhizium brunneum Conidia by Females of the Red Palm Weevil, Rhynchophorus ferrugineus, Suggests a New Mechanism for Prevention Practices.

Direct contact between the conidia of entomopathogenic fungi (EPF) and their host is a prerequisite to successful infection; the host can, therefore, be infected by both direct treatment and by transmission of fungal inoculum from infested surfaces. This unique characteristic makes EPF especially relevant for the control of cryptic insects. In the case of the red palm weevil (RPW) Rhynchophorus ferrugineus, the eggs and larvae are almost inaccessible to direct-contact treatment. The objective of the present study was to investigate the mechanism of conidia transmission from a treated surface to host eggs and larvae. Foam pieces infested with Metarhizium brunneum conidial powder, conidial suspension, or distilled water were used as a laying surface for RPW females. The number of eggs laid was not affected by the EPF treatments and ranged from 2 to 14 eggs per female. However, hatching rate and larval survival were significantly reduced in the conidial powder treatment, resulted in 1.5% hatching and no live larvae. In the conidial suspension treatment, 21% of laid eggs hatched, compared to 72% in the control treatment. In both M. brunneum treatments, females' proboscis, front legs and ovipositor were covered with conidia. The females transferred conidia in both treatments to the laying holes, reaching up to 15 mm in depth. This resulted in reduced egg-hatching rate and significant larval mortality due to fungal infection. The stronger effect on egg and larval survival using dry conidia seemed to result from better conidial adhesion to the female weevil in this formulation. In future studies, this dissemination mechanism will be examined as a prevention strategy in date plantations.

Pengembangan teknologi pengendalian hama utama kacang hijau menggunakan biopestisida

Hama merupakan salah satu kendala utama dalam upaya peningkatan produksi kacang hijau di Indonesia. Saat ini, pengendalian konvensional menggunakan pestisida sintetik kurang berhasil karena populasi dan kerusakan akibat hama masih tinggi. Penelitian ini bertujuan untuk mengembangkan biopestisida sebagai teknologi pengendalian hama utama kacang hijau yang dibandingkan dengan efikasi pestisida sintetik. Penelitian ini dilaksanakan menggunakan rancangan acak kelompok, lima perlakuan diulang lima kali. Perlakuan yang diuji, yaitu P1: aplikasi serbuk biji mimba (SBM), virus ulat grayak (Virgra) dari Spodoptera litura nuclear polyhedrosis virus, dan konidia cendawan entomopatogen Beauveria bassiana (BeBas) secara preventif; P2: aplikasi SBM, Virgra, dan BeBas berdasarkan ambang ekonomi (AE); P3: aplikasi pestisida sintetik sesuai jadwal; P4: aplikasi pestisida sintetik berdasarkan AE; dan P5: tanpa pengendalian (kontrol). Hasil penelitian menunjukkan bahwa hama yang berkembang adalah ulat grayak (Spodoptera litura (Fabricius)), kutukebul (Bemisia tabaci (Gennadius)), Empoasca sp., Megalurothrips usitatus (Bagnall), kepik coklat (Riptortus linearis (Fabricius)), kepik hijau (Nezara viridula (Linnaeus)), dan hama penggerek polong (Maruca testulalis (Geyer)). Aplikasi biopestisida SBM, Virgra, dan BeBas secara preventif dan aplikasi pestisida sintetik sesuai jadwal efektif menekan populasi hama utama kacang hijau, sedangkan aplikasi biopestisida dan pestisida sintetik berdasarkan AE tidak mampu menekan perkembangan populasi hama utama. Aplikasi biopestisida secara preventif maupun berdasarkan AE lebih aman terhadap kelangsungan hidup musuh alami, sedangkan aplikasi pestisida sintetik dapat membunuh musuh alami yang ada. Biopestisida SBM, Virgra, dan BeBas yang diaplikasikan secara preventif dan terintegrasi berpotensi sebagai inovasi teknologi pengendalian hama utama kacang hijau untuk menggantikan pestisida sintetik.

Optimised fermentation conditions and improved collection efficiency using dual cyclone equipment to enhance fungal conidia production

Methods for enhancing conidial yield and for harvesting pure fungal conidia of entomopathogenic fungi were investigated. Fermentation conditions (liquid-to-solid ratio, MgSO4·7H2O, incubation temperature, inoculum sizes, KNO3 and relative humidity) of Beauveria bassiana s.l. and Metarhizium anisopliae s.l. were optimised to increase the conidial yields that reached 11.2 mg/g and 24.5 mg/g, increases of 72% and 52% compared to the unoptimised yields of 6.5 mg/g and 16.1 mg/g, respectively. Three methods were compared for harvesting pure conidia of B. bassiana: dual cyclone equipment (DCE), sieving 200 and elution with 0.02% Tween-80 suspension. DCE performed the best, giving a conidial yield of 12.6 mg/g and 1.8 × 1010 conidia·g–1. To further enhance the harvest efficiency, response surface methodology combined with a Box–Behnken design was employed, and the conidial yield of B. bassiana reached 20.9 mg/g, a total increase of 221% compared to the original conditions. Under these optimised harvest parameters, the conidial yield of M. anisopliae rose to 42.2 mg/g, an increase of 162%. The conidia of B. bassiana and M. anisopliae harvested in this way were pure, with no mycelial fragments or substrate visible in microscopic images.

Carrying and Effect of Granulated Baits Formulated with Entomopathogenic Fungi among Atta sexdens rubropilosa Colonies (Hymenoptera: Formicidae)

The present study aimed to evaluate the carrying and effect of (dry) granulated baits containing conidia of entomopathogenic fungi among colonies of the leaf-cutting ant Atta sexdens rubropilosa in the laboratory. This bait type was chosen to facilitate its eventual commercial use. Two applications were performed: in the first, baits with 1x108 conidia/g were utilized while in the second employed concentrations 5 to 8.6 times greater. The baits were formulated with a citric pulp base, with 2 isolates of Beauveria bassiana, 1 of Paecilomyces lilacinus and 1 of Isaria fumosorosea. The following controls were utilized: (I) baits with sulfluramid insecticide, (II) without active ingredient, and (III) Acalypha spp. leaf discs. It was verified that the baits containing fungal conidia were rapidly carried to the nest interior in both applications and were rejected minimally. Thus, the (dry) granulated bait formulation appears to be an adequate vehicle for entomopathogenic fungi. At the doses and concentrations utilized, the fungi provoked only limited worker mortality, not killing the colonies. Given the rapid carrying and low rejection, a higher conidial dose per colony can, perhaps, kill them. Thus, it is inferred that all the isolates tested present potential as an agent to control colonies of leaf-cutting ants.

English

In vitro, the compatibility of eight vegetable oils, used as components in formulation, was studied in conidia of entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, based in three parameters that are been evaluated: germination rate, vegetative growth and conidiogenesis. Almond oil and gingelly oils at 1, 2 and 3% concentrations showed compatibility with B. bassiana as well as M. anisopliae. Mustard oil and eucalyptus oils at all three concentrations proved toxic to B. bassiana and M. anisopliae except at 1% concentration. Sunflower oil, olive oil, coconut oil and castor oils displayed compatibility with M. anisopliae and toxic to B. bassiana except olive oil and castor oil at 1% concentration. Conidiogenesis appear to be more affected than germination for the sample which displayed toxicity. Compatibility classification in to toxic, moderately toxic and compatible enabled assessment of the tested oils for use in formulations. Conidial formulations of B. bassiana and M. anisopliae with almond oil/olive oil/gingelly oil/castor oil were used for bioassaying against Spodoptera litura. All the four formulations displayed higher mortalities of the target pest compared to unformulated conidia.   Key words: Entomopathogenic fungi, compatibility, vegetable oils, concentrations, bioassay.

Biological Control of Aphid Using Fungal Culture and Culture Filtrates of Beauveria bassiana

Aphids are one of the most destructive pests in crop production such as pepper, cucumber, and eggplants. The importance of entomopathogenic fungi as alternative pest control agents is increasing. Conidia of entomopathogenic fungi are influenced by environmental conditions, such as temperature and relative humidity, and cause slow and fluctuating mortality. These factors have prevented wider application and use of biocontrol agents. For investigation of means of mitigation of such problems, we conducted bioassays with 47 fungal culture filtrates in order to evaluate the potential of secondary metabolites produced by entomopathogenic fungi for use in aphid control. Among 47 culture filtrates cultured potato dextrose broth, filtrate of Beauveria bassiana Bb08 showed the highest mortality (78%) against green peach aphid three days after treatments. Filtrate of Bb08 cultured in Adamek’s medium showed higher toxicity as 100% to third instar nymphs of the aphid compared with seven other filtrates cultured in different broths amended with colloidal chitin or oil. The culture filtrates and fungal cultures from media amended with colloidal chitin or oil had lower control efficacies than filtrates without these additives in three different media. These results indicate that the fungal culture fluid or culture filtrate of B. bassiana Bb08 cultured in Adamek’s medium has potential for development as a mycopesticide for aphid control.

Identification of proteases produced by entomopathogenic fungi Beauveria bassiana (Bals) Vuill. strain CG432 previously activated in coffee berry borer alive (Hypothenemus hampei).

Conidia of entomopathogenic fungi can penetrate the insect exoskeleton both by mechanic action of the germinative tube and by production of multiple proteases, chitinases, and lipases in response to the composition of the insect cuticle. Therefore the purpose of this work was to produce, to purify, and to characterize the structure of the proteases produced by the fungus Beauveria bassiana CG432, previously activated in adults of coffee berry borer ( Hypothenemus hampei) grown under submerged culture conditions. A suspension containing 106 activated conidia/mL was inoculated to a culture medium at 28oC and 150 rpm for 3 days. Protease extracts, were obtained by centrifugation at 8000 g for 20 minutes, were fractionated and were concentrated by ultrafiltration using controlled porosity membranes of 100 kDa and 3 kDa, respectively. Gel filtration chromatography on Sephadex G-100 isolated one protein peak (peak II), which contained 56% of residues of the aminoacid aspartic acid, characterized by HPLC using an ODS-C18 reverse phase column. The peak protein showed specific activity 43 times superior when compared to the free-cell extract in bovine serum albumin, subtilisin-like protease activity, and a single protein band reveled by Brilliant Coomassie Blue on a gelatin zimogram PAGE electrophoresis, by native conditions. The homogeneity of peak II was confirmed by revelation of a single band during determination of the isoelectric pH 4.5, but molecular mass determination by PAGE-2D electrophoresis showed 2 bands of 23 and 26 kDa. Proteases were characterized as serine proteases with cysteine residues important to activity, since they had been inhibited by phenylmethylsulphonyl fluoride and p -chlormercuricbenzoic acid. Peak II proteases showed Km of 4x10-4 on subtilisin-like substrate.

Primary and secondary uptake of conidia of entomopathogenic fungi and the effect of oxybenzone as a chemical sunlight protector

Primary and secondary uptake of conidia of entomopathogenic fungi and the effect of oxybenzone as a chemical sunlight protector

Development of a Spray-drying Technique for Submerged Spores of Entomopathogenic Fungi

The stability of blastospores or submerged conidia of entomopathogenic fungi is one of the key problems associated with the practical use of fungal biopreparations. A spray-drying technique was developed which allows the drying of blastospores and/or submerged conidia, here called submerged spores, without a significant loss of viability and with only a slight delay in eY cacy. The method was tested successfully using the fungal species Metarhizium anisopliae , M. flavoviride, Beauveria bassiana and Paecilomyces fumosoroseus . For all experiments, a laboratory spray dryer was used. The optimum inlet and outlet temperatures were found to be 64 2oC and 48 2oC respectively. The best protective agent for submerged spores was skimmed milk powder at concentrations of 10 or 20%. The addition of sugars, especially 2.5% sugar-beet syrup, slightly improved the viability after spray-drying. Submerged spores of eight isolates of M. anisopliae, M. flavoviride, B. bassiana and P. fumosoroseus were suspended in 20% skimmed milk powder and 2.5% sugar-beet syrup, and spray-dried. Germination rates of about 90% were achieved, which were comparable to those of freshly produced submerged spores. Finally, the virulences of spray-dried submerged spores of M. anisopliae (Ma 97) and M. flavoviride (Mfl 5) were tested in bioassays using third- and fourth-instar nymphs of the African locust, Locusta migratoria . In contrast to M. flavoviride , there was no significant diVerence in the median lethal time between spray-dried and fresh submerged spores of M. anisopliae . The investigations demonstrated that spray-drying is possible to preserve sensitive submerged spores of entomopathogenic fungi.