Among the prospective biocontrol agents, the saprophytic filamentous fungus
is an excellent necrotrophic mycoparasite of numerous plant pathogenic fungi. However, its commercial development has been ...hampered by mass production difficulties during solid-state fermentation. Conversely, the submerged liquid fermentation shortens the cultivation time while increasing yields of fungal propagules. However, this method has been overlooked for
. In this work, we investigated the impact of liquid pre-culture inoculum on the spore production by the two-stage fermentation process using rice grains in comparison to the traditional solid-state fermentation. In parallel, we studied the submerged cultivation of
by manipulating carbon-to-nitrogen (C:N) ratio and nitrogen source, with the further optimization of spore production in a benchtop bioreactor. Additional bioassays included assessing the bioactivity of water-dispersible microgranules (that contained a submerged conidia) against the whitefly (
biotype B) and
(causal agent of the white mold). Our results showed a maximum concentration of 1.1 × 10
conidia/g-dry-matter after 7 days of cultivation by two-stage fermentation process. The liquid fermentation yielded 1.4 × 10
submerged conidia/ml after 7 days using a medium with a 50:1 C:N ratio, and it also induced the production of microsclerotia (MS) up to 1.35 × 10
/ml within 6 days with 10:1 C:N ratio; both media were supplemented with dextrose monohydrate and soybean meal. The fermentation batches carried out in a benchtop bioreactor with medium 50:1 C:N ratio and amended with soybean meal rendered a production peak on the fourth day, corresponding to 1.11 × 10
conidia/ml and 4.35 × 10
colony forming units (CFU)/ml. Following air-drying, the conidia production from air-dried microgranules of
biomass was estimated at 3.4 × 10
conidia/g of formulated product upon re-hydration for 7 days. Both submerged conidia and MS of
inhibited 100% germination of
sclerotia by direct parasitism. The air-dried submerged conidia exhibited a suppressive activity on sclerotia (88% mycoparasitism) and early whitefly nymphs (76.2% mortality) that rendered LC
values of 3.2 × 10
CFU/g soil and 1.5 × 10
CFU/ml, respectively. Therefore, the submerged liquid culture of
may offer a feasible and cost-effective method for its large-scale production, alleviating critical constraints to their commercial use while providing an additional tool for management of
and
.
Beauveria bassiana
is a cosmopolitan entomopathogenic fungus that can infect over 1000 insect species. During growth inside the host,
B. bassiana
transitions from hyphal to yeast-like unicellular ...growth as blastospores. Blastospores are well suited as an active ingredient in biopesticides due to their ease of production by liquid fermentation. Herein, we investigated the impact of hyperosmotic growth environments mediated by ionic and non-ionic osmolytes on two strains of
B. bassiana
(ESALQ1432 and GHA) relevant to growth morphology, blastospore production, desiccation tolerance, and insecticidal activity. Polyethylene glycol (PEG200) increased osmotic pressure in submerged cultures leading to decreased blastospore size but higher blastospore yields for one strain. Morphologically, decreased blastospore size was linked to increased osmotic pressure. However, smaller blastospores from PEG200 supplemented cultures after air-drying exhibited delayed germination. Ionic osmolytes (NaCl and KCl) generated the same osmotic pressure (2.5–2.7 MPa) as 20% glucose and boosted blastospore yields (> 2.0 × 10
9
blastospores mL
−1
). Fermentation performed in a bench-scale bioreactor consistently promoted high blastospore yields when using NaCl (2.5 MPa) amended media within 3 days. Mealworm larvae (
Tenebrio molitor
) were similarly susceptible to NaCl-grown blastospores and aerial conidia in a dose-time-dependent manner. Collectively, these results demonstrate the use of hyperosmotic liquid culture media in triggering enhanced yeast-like growth by
B. bassiana.
Understanding the role of osmotic pressure on blastospore formation and fitness will hasten the development of viable commercial fungal biopesticides.
Key points
• Osmotic pressure plays a critical role in submerged fermentation of B. bassiana.
• Ionic/non-ionic osmolytes greatly impact blastospore morphology, fitness, and yield.
• Desiccation tolerance and bioefficacy of blastospores are affected by the osmolyte.
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•Higher virulence against nymphs was attained with B. bassiana and I. fumosorosea.•None of the B. bassiana and I. fumosorosea isolates were effective against eggs.•Whitefly adults are ...highly susceptible to I. fumosorosea isolates.•Selection was based on virulence and sporulation on rice and nymphal cadavers.
The virulence of five isolates of Beauveria bassiana, five of Isaria fumosorosea and four of Lecanicillium muscarium from Brazil was determined on whitefly Bemisia tabaci biotype B lifestages on bean leaves under laboratory conditions. The conidial yield (on cadavers or parboiled rice), surface hydrophobicity and enzyme activity were also determined. The isolates of B. bassiana and I. fumosorosea were the most virulent against nymphs (71–86% mortality within 8d), with LT50 values ranging from 3 to 4d after treatment with 107conidia/mL (150conidia/mm2). Spore production on nymph cadavers reached 4–8×105conidia/insect. The L. muscarium isolates demonstrated low virulence toward nymphs. After spraying eggs with 1×108conidia/mL (1674conidia/mm2) of B. bassiana and I. fumosorosea, most nymphs hatched, but then 40–70% of these nymphs were infected by indirect exposure of conidia on the leaves. Adults exposed to treated leaf disks (150conidia/mm2) were more susceptible to I. fumosorosea than to B. bassiana. The enzyme activity (Pr1) and the relative conidial surface hydrophobicity were not correlated with any virulence parameter measured for either B. bassiana or I. fumosorosea. In addition, the highest conidial yields on parboiled rice using solid-state fermentation (4.9–11.4×109conidia/g) were achieved by isolates of I. fumosorosea CG1228 and B. bassiana CG1229. I. fumosorosea CG1228 was highly virulent against whitefly nymphs and adults as well as attained high spore production on insect cadavers and parboiled rice. Our results indicate that I. fumosorosea CG1228 has desirable attributes for the development of a mycoinsecticide against B. tabaci biotype B.
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•We have developed a cost-effective liquid medium for blastospore production.•Cottonseed flour is a suitable nitrogen source for rapid production of blastospores.•B. bassiana and I. ...fumosorosea blastospores are desiccation tolerant and storage stable.•Blastospores are more virulent toward whitefly nymphs than aerial conidia.
A major constraint to the commercial use of fungal biocontrol agents is the availability of low-cost production media and processes. Previous attempts in producing Beauveria blastospores using liquid culture fermentation processes required long fermentation times (6–8days) and produced cells that had poor survival after desiccation and storage. In this study, isolates of Beauveria bassiana and Isaria fumosorosea were evaluated for blastospore yield, desiccation tolerance, storage stability, and biocontrol efficacy using fermentation media containing acid hydrolyzed casein or cottonseed flour as the nitrogen source. Cultures of B. bassiana and I. fumosorosea grown in media containing cottonseed flour produced high blastospore concentrations (>1×109mL−1) after 3days which is comparatively less expensive nitrogen source than acid hydrolyzed casein. The resultant air-dried blastospores (<3% moisture) of all fungal isolates survived drying (61–86% viability), irrespective of the nitrogen source tested. Storage stability at 4°C varied with nitrogen source and fungal strain. Air-dried blastospores of B. bassiana strains showed half-lives >14months in contrast to 9.2–13.1months for I. fumosorosea. Blastospores of B. bassiana and I. fumosorosea killed Bemisia tabaci whitefly nymphs faster and required lower concentrations compared with aerial conidia. Our findings support the use of liquid culture fermentation as a cost-effective process to rapidly produce high yields of stable and infective blastospores of either B. bassiana or I. fumosorosea. These results support further evaluation of blastospore sprayable formulations for the control of soft-bodied insects.
O conhecimento das estruturas anatômicas da semente e do embrião podem contribuir para a compreensão do processo germinativo e na interpretação de testes de germinação e vigor. Dessa forma, ...objetivou-se descrever morfologicamente os diásporos (sementes com o endocarpo aderido) e plântulas de Livistona chinensis. Foram coletados dados dos parâmetros biométricos dos diásporos (largura, comprimento, peso de mil diásporos e número de diásporos kg-1), bem como foram realizadas mensurações do teor de água destes. Foram semeados 100 diásporos em bandejas de plástico contendo como substrato a vermiculita®. Para a descrição da morfologia do diásporo e do crescimento inicial da plântula, as avaliações foram realizadas acompanhando o processo germinativo, a partir da primeira semana, em cada estádio de desenvolvimento, até a emissão do primeiro eófilo. As descrições morfológicas foram baseadas em terminologia específica e esquematizados com auxílio de câmara clara acoplada ao estereomicroscópio. A germinação é caracterizada como sendo do tipo remota tubular. Os diásporos apresentam forma oval e as sementes são albuminosas, com endosperma rígido, embrião lateral periférico, cônico e pouco diferenciado. A primeira folha é simples e lanceolada, nervação paralela, composta por nervuras largas, dispostas longitudinalmente.
Knowledge of the anatomical structures of the seed and embryo can contribute to understanding the germination process and interpreting germination and vigor tests. Thus, the objective of this study ...was to describe morphologically the diaspores (seeds with adhered endocarp) and seedlings of Livistona chinensis. Biometric parameters of the diaspores (width, length, weight of one thousand diaspores, and number of diaspores kg-1) were collected, and their water content was measured. One hundred diaspores were sown in plastic trays containing vermiculite as a substrate. To describe the morphology of the diaspore and the initial seedling growth, evaluations were performed by monitoring the germination process from the first week, at each stage of development, until the emergence of the first eophyll. Morphological descriptions were based on specific terminology and schematized with the aid of a clear camera attached to a stereomicroscope. Germination is characterized as being of the remote tubular type. The diaspores have an oval shape with an average length of 16.37 mm and an average width of 10.25 mm, and the seeds are albuminous with a rigid endosperm, peripheral lateral cone-shaped embryo, and little differentiation. The first leaf is simple and lanceolate, with parallel venation, composed of broad veins arranged longitudinal.
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.
Several non-ionic surfactants can be used to enhance insecticidal performance of various bio-based products and agrochemicals. In this study, we describe the toxicity of four commercial non-ionic ...surfactants against immature Bemisia tabaci (Gennadius) biotype B whiteflies along with their spreading ability. Results revealed that trisiloxane-based surfactants (Break-thru® and Silwet® L-77) exhibited the highest toxicity to 1st–2nd (early) and 3rd–4th (late) instar nymphs as well as the greatest wetting performance. We also investigated whether sub-lethal concentrations of Break-thru® and Silwet® L-77 improved the insecticidal efficacy of the fungal entomopathogens Beauveria bassiana (Balsamo) Vuillemin (strain CG1229) and Isaria fumosorosea Wise (strain CG1228) conidial suspensions against whiteflies. Germination of hydrophobic conidia of both isolates were unaffected by these surfactants over the range 100–1000 ppm. The combinations of both fungi with trisiloxane carriers significantly increased nymphal mortality with mostly additive and synergistic effects on early and late nymphs, respectively. In screenhouse trials, both fungi (10⁷ conidia ml⁻¹) mixed with 200 ppm of Silwet® L-77 significantly improved effectiveness against early nymphs (72–74 % mortality) compared with controls treated with water and Silwet® L-77 alone. In addition, reduced volume rates of I. fumosorosea in Silwet® L-77 (equivalent to 100 l ha⁻¹) were at least as effective against late nymphs as higher volume rates (200 l ha⁻¹) at equivalent conidial doses. Our findings underline the compatibility and enhanced activity of silicon-based surfactants with B. bassiana and I. fumosorosea for use in integrated whitefly management programs.
Incidência de patógenos e vigor de sementes de milho doce submetidas a danos mecânicos Gomes Junior, Francisco Guilhien(Universidade de São Paulo Escola Superior de Agricultura Luiz de Queiroz Departamento de Produção Vegetal); Timóteo, Tathiana Silva(Universidade de São Paulo Escola Superior de Agricultura Luiz de Queiroz Departamento de Produção Vegetal); Kobori, Nilce Naomi(Universidade de São Paulo Escola Superior de Agricultura Luiz de Queiroz Departamento de Produção Vegetal) ...
Summa phytopathologica,
2009, Letnik:
35, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Com o objetivo de avaliar a incidência de patógenos e o vigor de sementes de milho doce de diferentes classes de tamanhos submetidas a diferentes níveis de danos mecânicos, sementes do híbrido ...'SWB551' Dow AgroSciences® foram classificadas em peneiras com crivos de diferentes tamanhos e formas (RG, RM1, RM2 e RP com crivos circulares de 8,7; 7,9; 7,1 e 6,4 mm de diâmetro, respectivamente, e CG, CM e CP com crivos oblongos com dimensões de 8,7 x 19,0, 7,9 x 19,0, 6,4 x 19,0 mm, respectivamente), submetidas a impactos contra uma placa metálica de maneira que fossem obtidos tratamentos com diferentes intensidades de danos mecânicos (0, 1, 3, 5 e 7 impactos) e armazenadas por 5 meses em ambiente com temperatura de 20ºC e 50-60% de umidade relativa do ar. Foi utilizado o delineamento inteiramente casualizado, com quatro repetições, perfazendo um fatorial 7 x 5 (7 peneiras x 5 intensidades de danos), totalizando 35 tratamentos. Foram realizadas as avaliações de teor de água das sementes, teste de danos mecânicos (tintura de iodo), teste de germinação, teste de frio e teste de sanidade. Foram evidenciadas variações nos níveis de incidência de patógenos entre as sementes de milho doce com diferentes tamanhos e formas. O aumento da intensidade dos danos mecânicos não favoreceu o aumento da incidência de patógenos nas sementes, mas reduziu o vigor das mesmas.
The objective of this study was to evaluate pathogen incidence and vigor in different size classes of sweet corn seeds, after mechanical damage. Hybrid seeds 'SWB551' Dow AgroSciences® were classified by sieves with different sizes and forms (RG, RM1, RM2 and RP, with circular sieves of 8.7, 7.9, 7.1 and 6.4 mm of diameter, respectively, and CG, CM and CP with oblong sieves with dimensions of 8.7 x 19.0, 7.9 x 19.0, 6.4 x 19.0 mm, respectively). Seeds were submitted for impacts against a metallic plate in order to have different intensities of mechanical damage (0, 1, 3, 5 and 7 impacts) and stored at the temperature of 20ºC and 50-60% of relative humidity. The experimental design was a completely randomized with four repetitions, performing a factorial 7 x 5 (7 sieves and 5 damages intensities), totalizing 35 treatments. Seeds water content, mechanical damage (iodine dyeing), germination test, cold test and seed health test were evaluated. Variations in the incidence of pathogens among seeds with different sizes and forms were evidenced. Increasing mechanical damage did not favor increasing of pathogens incidence in seeds; however, it caused reduction on seed vigor.
A Palmeira-leque-da-China é uma espécie bastante ornamental e muito utilizada em paisagismo. O objetivo deste trabalho foi verificar a influência de diferentes temperaturas e regimes de luz na ...germinação de sementes desta espécie. O delineamento experimental adotado foi inteiramente casualizado, em esquema fatorial 6 x 2 (seis temperaturas – constantes de 20, 25, 30, 35ºC e alternadas de 20-30 e 25-35ºC – associadas a dois regimes de luz – 8 horas de luz ou escuro contínuo). O experimento foi realizado em câmara de germinação do tipo BOD (Biochemical Oxygen Demand), com quatro repetições de 25 sementes, em caixas gerbox transparentes ou pretas, contendo vermiculita. As avaliações foram realizadas diariamente, considerando-se germinadas as sementes que apresentavam a protrusão do botão germinativo. Foi constatada alta porcentagem de germinação – 96 a 99% – independentemente da temperatura e do regime de luz, e também maior índice de velocidade de germinação na temperatura alternada de 25-35ºC, com regime de 8 horas de luz.
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