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Comparative analysis of Beauveria bassiana submerged conidia with blastospores: yield, growth kinetics, and virulence
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Growers rely on synthetic insecticides to control the boll weevil throughout the reproductive cotton stage. An average of 19.6 insecticide applications (range: 10 to 30) for control of boll weevil were found in a survey with growers in the Brazilian Cerrado, covering an area of 494,100 hectares of cotton. Twenty-one insecticides were applied, with 64.8% of the applications made with malathion, fipronil, carbosulfan, and thiamethoxam + lambda-cyhalothrin. These four insecticides were used by 100, 76, 70, and 62% of the growers, with respectively 7.2, 2.1, 1.8, and 1.6 applications. Growers classified their boll weevil control achieved into four categories (fair, good, very good, or excellent), without correlation between these categories with the number of insecticide applications. Control of cotton regrowth and volunteer cotton plants were the major obstacles for effective boll weevil management, followed by the low efficacy of insecticides. Five registered insecticides to spray cotton against other pests than boll weevil were enlisted by growers with potential for recommendation. A boll weevil standard population for susceptibility was assayed with 27 insecticides and the results presented within a failure risk quotient (FRQ). The FRQ of eight, six, and 13 of the 27 tested insecticides was high, intermediate, and low, respectively. The high FRQ included 7 of 10 pyrethroid formulations, pymetrozine, and methomyl. On the opposite end, fipronil had the lowest FRQ value.
p> Background. Resistance to an insecticide is what determines its effectiveness and the lack of information limits effectiveness in the control of the boll weevil in cotton crops. Objective. The objective of the study was to determine the susceptibility of the boll weevil ( Anthonomus grandis Boheman) to malathion in the Laguna, Mexico. Currently, this pest is considered as the one with the highest economical impact on cotton cultivation in the region. Methodology. Three bioassays were carried out, one in the state of Coahuila in the 2017 agricultural season and in the 2018 and 2019 seasons in the states of Coahuila and Durango. Boll weevils reared in the laboratory, as well as those collected in the field were used to obtain DL50 and DL95. The mortality data was analyzed using the Probit analysis. Results. The mean lethal dose after 48 hours in the three agricultural cycles fluctuated between 0.19 and 0.39 µg/µl for adults raised in the laboratory and the data observed for adults collected in the field was between 0.37 and 0.44 µg/µl. Implications. Periodic bioassays are suggested to detect populations resistant to malathion. Conclusion. It was determined that the boll weevil populations in the Laguna Region were susceptible to malathion, since the mean lethal dosage for the 3 agricultural seasons was < 1 µg/µl.</p
The present research addressed spray-drying and air-drying techniques applied to Metarhizium robertsii blastospores to develop wettable powder (WP) formulations. We investigated the effect of co-formulants on blastospore viability during drying and assessed the wettability and stability of formulations in water. The effect of oxygen-moisture absorbers was studied on the shelf life of these formulations stored at 26 °C and 4 °C for up to 90 days. Additionally, we determined the virulence of the best spray-dried and air-dried formulations against the corn leafhopper Dalbulus maidis. While sucrose and skim milk played an essential role as osmoprotectants in preserving air-dried blastospores, maltodextrin, skim milk, and bentonite were crucial to attain high cell survival during spray drying. The lowest wettability time was achieved with spray-dried formulations containing less Ca-lignin, while charcoal powder amount was positively associated with formulation stability. The addition of oxygen-moisture absorbers inside sealed packages increased from threefold to fourfold the half-life times of air-dried and spray-dried formulations at both storage temperatures. However, the half-life times of all blastospore-based formulations were shorter than 3 months regardless of temperature and packaging system. Spray-dried and air-dried WP formulations were as virulent as fresh blastopores against D. maydis adults sprayed with 5 × 10⁷ blastospores mL⁻¹ that induced 87.8% and 70.6% mortality, respectively. These findings bring innovative advancement for M. robertsii blastospore formulation through spray-drying and underpin the importance of adding protective matrices coupled to oxygen-moisture absorbers to extend cell viability during either cold or non-refrigerated storage.
Graphic abstract
Key points
• Cost-effective wettable powder formulations of M. robertsii blastospores were developed.
• Bioefficacy of formulations against the corn leafhopper was comparable to fresh blastospores.
• Cold storage and dual oxygen-moisture absorber are critical for extended shelf life.
BACKGROUND
Insecticide resistance in arthropods is an inherited trait that has become a major cause of insect pest control failure. Monitoring the level of susceptibility and characterization of the type of resistance of key pest species aims to determine the risk of resistance selection in time to take action to mitigate control failures. Seven populations of the boll weevil, Anthonomus grandis grandis, collected from cotton fields in the Semiarid and Cerrado areas of Brazil, were screened for their resistance to malathion and beta‐cyfluthrin, insecticides widely recommended for control of boll weevil and other pests.
RESULTS
The levels of adult mortality were variable for beta‐cyfluthrin (0–82%) but invariant (100%) for malathion. Bioassays of concentration‐mortality were used to determine lethal concentrations (LCs) for each insecticide. The LC‐values corroborate the lack of resistance to field rates of malathion but high levels of resistance to beta‐cyfluthrin from 62.7‐ to 439.7‐fold. Weevils resistant to beta‐cyfluthrin were found through genome sequencing to possess a kdr mutation through the L1014F substitution in the voltage gated‐sodium channel gene.
CONCLUSIONS
This study found boll weevil resistance to beta‐cyfluthrin to be not mediated by carboxylesterases, but with cross‐resistance to DDT and carbaryl, and kdr mutation as the major mechanism of the resistance in our samples. Caution is recommended in further use of beta‐cyfluthrin against boll weevil due to potential resistance. Monitoring studies using other boll weevil populations are recommended to determine the geographic pattern and extent of pyrethroid resistance. © 2021 Society of Chemical Industry.
The fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a highly polyphagous plant pest that can severely impact yields of several agricultural crops. Understanding the economic impact and management thresholds for FAW across a variety of crop commodities is crucial for effective management. Evaluating the peer-reviewed and grey literature, we compiled global data on: (1) yield losses reported as a result of FAW infestations, (2) the relationship between FAW pressure/density and reported yield loss, and (3) current known economic injury levels, economic thresholds and action thresholds. We identified 71 references that reported yield losses from FAW infestation, with a total of 888 separate yield loss entries. The majority of research quantifying yield losses and the relationship between pest pressure and yield has focused on maize, sorghum, and cotton, with some evidence for sweet corn, bermudagrass, and rice. Yield loss varied between management strategies, with genetically modified and/or insecticide treated crops typically retaining higher yields. Most studies investigating the relationship between FAW density and yield loss across different crops have focused on early and mid FAW larval instars and on vegetative through to reproductive plant growth stages, with minimal research on both late larval instars and on plant seedlings. Economic thresholds were not reported in the literature. The reporting of economic injury levels and action thresholds varied significantly both between and within crops, highlighting the need for a standardised approach when measuring FAW pressures or densities that elicit management responses.
The first detection of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in Brazil pointed to a gloomy future for South American agriculture in the 2012/2013 harvest, and insecticide sales increased exponentially during 2013 and 2014. However, after this outbreak, the population of this insect returned to manageable levels. This study documents data on H. armigera moth collection using light traps during the outbreak in two locations: (a) western region of the state of Bahia, including population levels of H. armigera and main noctuid pests during the big outbreak (February and March 2013); (b) Federal District, including monthly data for five growing seasons (July 2012-June 2017). During the outbreak, an average of 527.3 moths were collected per trap (cotton crop, March) in the western region of Bahia each night, while the maximum average in the Federal District was 75.4 moths (January). The number of moths decreased in the following growing seasons, and in October 2016, the maximum number was 2.8 moths per trap. Aspects related to the variations of H. armigera and Helicoverpa zea (Boddie) populations, including the study of phenology and relationship with climatic factors and Oceanic Niño Index, were analyzed and discussed. All aspects are approached to contribute to the management of lepidopteran pests, especially H. armigera, in the productive systems of the Brazilian Cerrado.
The cosmopolitan entomopathogenic and root endophytic fungus Metarhizium robertsii has a versatile lifestyle and during liquid fermentation undergoes a dimorphic transformation from hyphae to conidia or microsclerotia, or from hyphae to blastospores. In all cases, these processes are mediated by environmental and nutritional cues. Blastospores could be used in spray applications to control arthropod pests above ground and may serve as an attractive alternative to the traditional solid-grown aerial conidial spores of Metarhizium spp. found in commercial products. Nitrogen is a vital nutrient in cell metabolism and growth; however, it is the expensive component in liquid cultures of entomopathogenic fungi. Our goals in this study were to optimize nitrogen sources and titers for maximum production of M. robertsii blastospores cultured in shake flasks at highly aerated conditions and to further determine their virulence against the corn leafhopper Dalbulus maidis, an important vector of serious pathogens in maize crops worldwide. Our fermentation studies revealed that the low-cost corn steep liquor (CSL) was the most suitable nitrogen source to improve blastospore growth in M. robertsii. The growth kinetic assays determined the optimal titer of 80 g L−1 and a yield up to 4.7 × 108 cells mL−1 within 5 days of cultivation (3 days preculture and 2 days culture), at a total cost of US$0.30 L−1. Moreover, the blastospore growth kinetic was strongly dependent on glucose and nitrogen consumptions accompanied by a slight drop in the culture pH. Insect bioassays evidenced a high virulence of these blastospores, either as dried or fresh cells, to D. maidis adults fed on maize plants. Our findings provide insights into the nutritional requirements for optimal and cost-efficient production of M. robertsii blastospores and elucidate the potential of blastospores as an ecofriendly tool against the corn leafhopper.
BACKGROUND
The use of Bt plants has been the main strategy for controlling the fall armyworm Spodoptera frugiperda (J. E. Smith) in Brazil. However, many resistance cases were already registered. The resistance of S. frugiperda to the Vip3Aa20 protein was recently characterized under laboratory conditions but it is still efficient under field conditions. Here, resistance monitoring studies were conducted using phenotypic (purified protein and Bt maize leaves) and genotypic (F1 and F2 screen) methods to support insect resistance management (IRM) programs and preserve Vip3Aa20 technology on maize.
RESULTS
Phenotypic monitoring with purified protein showed two populations significantly different from the susceptible strain on the second crop season in 2016. This number increased for the first and second crop seasons in 2017 in several regions. The genotypic monitoring estimated a mean frequency of the resistance allele of 0.0027 for the F1 screen and 0.0033 for the F2 screen. Three new resistant strains to Vip3Aa20 were selected from F2 screen assays. Complementation tests on these new resistant strains were positive with the previous resistant strain.
CONCLUSION
Here we showed that the resistance allele of S. frugiperda to Vip3Aa20 protein is widely distributed in maize‐producing regions in Brazil and its frequency increases throughout crop seasons. © 2019 Society of Chemical Industry
BACKGROUND
Understanding the genetic basis of insect resistance to insecticides can help to implement insecticide resistance management (IRM) strategies. In this study, we selected a strain of Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) resistant to chlorantraniliprole using the F2 screen method, characterized the inheritance of resistance and evaluated patterns of cross‐resistance to other diamide insecticides.
RESULTS
The chlorantraniliprole‐resistant strain (Chlorant‐R) was selected from a field‐collected population with an estimated allele frequency of 0.1316. The estimated median lethal concentration (LC50) values were 0.011 and 2.610 µg a.i. cm–2 for the susceptible (Sus) and Chlorant‐R strains, respectively, resulting in a resistance ratio (RR) of 237‐fold. The LC50 values of the reciprocal crosses were 0.155 and 0.164 µg a.i. cm–2, indicating that resistance is autosomally inherited. Resistance was characterized as incompletely recessive and monogenic at concentrations close to the recommended field rates of chlorantraniliprole. Survival of the resistant strain and heterozygous larvae in maize plants treated at the field rate was ∼ 60 (similar to the susceptible strain on untreated maize) and 15%, respectively. The Chlorant‐R strain showed low cross‐resistance to cyantraniliprole (RR ∼ 27‐fold) and very high cross‐resistance to flubendiamide (RR > 42 000‐fold).
CONCLUSIONS
The frequency of chlorantraniliprole resistance allele was moderately high in a field‐collected population of S. frugiperda. The inheritance of chlorantraniliprole resistance was characterized as autosomal, incompletely recessive and monogenic. S. frugiperda showed cross‐resistance to other diamide insecticides. These results provide essential information for the implementation of IRM programs to preserve the useful life of diamide insecticides for controlling S. frugiperda in Brazil. © 2019 Society of Chemical Industry
Biorational insecticides are being increasingly used in integrated pest management programs. In laboratory bioassays, the pathogenicity of blastospores and conidia of the entomopathogenic fungus Metarhizium brunneum ARSEF 4556 was evaluated against larvae of three mosquito species. Three propagule concentrations (1 × 106, 1 × 107, and 1 × 108 spores ml - 1) were used in the bioassays. Results showed that Aedes aegypti had lower survival rates when exposed to blastospores than when exposed to conidia, whereas the converse was true for Culex quinquefasciatus larvae. Anopheles stephensi larvae survival rates were similar when exposed to blastospores and conidia, except at the higher doses, where blastospores were more virulent. Several assays showed little difference in mortalities when using either 1 × 107 or 1 × 108 spores ml - 1, suggesting a threshold above which no higher control levels or economic benefit would be achieved. When tested at the lowest dose, the LT50 of Cx. quinquefasciatus using blastospores, wet conidia, and dry conidia was 3.2, 1.9, and 4.4 d, respectively. The LT50 of Ae. aegypti using blastospores, wet conidia, and dry conidia was 1.3, 3.3, and 6.2 d, respectively. The LT50 of An. stephensi using blastospores, wet conidia, and dry conidia was 2.0, 1.9, and 2.1 d, respectively. These observations suggest that for optimized control, two different formulations of the fungus may be needed when treating areas where there are mixed populations of Aedes, Anopheles, and Culex.
flexsurv
is an R package for fully-parametric modeling of survival data. Any parametric time-to-event distribution may be fitted if the user supplies a probability density or hazard function, and ideally also their cumulative versions. Standard survival distributions are built in, including the three and four-parameter generalized gamma and F distributions. Any parameter of any distribution can be modeled as a linear or log-linear function of covariates. The package also includes the spline model of Royston and Parmar (2002), in which both baseline survival and covariate effects can be arbitrarily flexible parametric functions of time. The main model-fitting function, flexsurvreg, uses the familiar syntax of survreg from the standardsurvivalpackage (Therneau 2016). Censoring or left-truncation are specified in 'Surv' objects. The models are fitted by maximizing the full log-likelihood, and estimates and confidence intervals for any function of the model parameters can be printed or plotted.flexsurvalso provides functions for fitting and predicting from fully-parametric multi-state models, and connects with themstatepackage (de Wreede, Fiocco, and Putter 2011). This article explains the methods and design principles of the package, giving several worked examples of its use.
Insects are the dominant animals in the world, with more than one million described species. The vast majority of insects are innocuous or beneficial to humans, but a small percentage are pests that require a significant amount of our time, effort, and funds to reduce their negative effects on food production and our health and welfare. One environmentally acceptable method to control these insect pests is to use pathogens. The study of pathogens infecting insects is referred to as “insect pathology”. Insect pathology is the study of anything that goes wrong with an insect and, therefore, includes non-pathogenic and pathogenic causes. The present focus is on pathogens that can be used as microbial control agents of insects. Here, the basic principles in insect pathology including the microorganisms that cause diseases, their classification and phylogeny, portal of entry, infectivity, pathogenicity and virulence, course of disease, Koch’s postulates, and
diagnosis are covered.
Understanding of the ecology of fungal entomopathogens has vastly increased since the early 1800's, but remains challenging. The often complex interactions between pathogen and host are being unravelled through eloquent research and the importance of the often subtle interactions, in determining the success or failure of biological control, cannot be underplayed. The realm of ecology is vast and deciphering insect-fungal pathogen interactions within an ecological context will take us on voyages beyond our imagination. This book brings together the work of renowned scientists to provide a synthesis of recent research on the ecology of fungal entomopathogens exploring host-pathogen dynamics from the context of biological control and beyond. Dr. Helen Roy leads zoological research in the Biological Records Centre at the NERC Centre for Ecology & Hydrology, UK. The focus of her research is insect community interactions with particular emphasis on the effects of environmental change. She has been working on the ecological interactions between fungal entomopathogens and their hosts for 15 years; this continues to be a source of fascination. She has been an associate editor of BioControl since 2006. Dr. Dave Chandler is an insect pathologist at the University of Warwick, UK. He has studied entomopathogenic fungi for just over 20 years. He has particular interests in entomopathogenic fungi as biocontrol agents of horticultural crops, fungal physiology and ecology, and the pathogens of honeybees. Dr. Mark Goettel is an insect pathologist at the Lethbridge Research Centre of Agriculture & Agri-Food Canada, specializing in the development of fungal entomopathogens as microbial control agents of insects. In addition to this research, he has been extensively involved in the review and revision of the regulations for registration of microbial control agents and has addressed regulatory and safety issues at the international level. He is currently President of the Society for Invertebrate Pathology and has been Editor-in-Chief of Biocontrol Science & Technology since 2000. Dr. Judith K. Pell heads the Insect Pathology Group in the Department for Plant and Invertebrate Ecology at Rothamsted Research, UK. She leads research on the ecology of fungal entomopathogens, to elucidate their role in population regulation and community structure and to inform biological control strategies. Specifically: intraguild interactions; the relationships between guild diversity, habitat diversity and ecosystem function; pathogen-induced host behavioural change. Dr. Eric Wajnberg is a population biologist specialising in behavioural ecology, statistical modelling and population genetics. He is also an expert in biological control, with more than 20 years experience of working with insect parasitoids. He has been the Editor in Chief of BioControl since 2006. Dr. Fernando E. Vega is an entomologist with the United States Department of Agriculture, Agricultural Research Service, in Beltsville, Maryland, USA. He conducts research on biological methods to control the coffee berry borer, the most important insect pest of coffee throughout the world. He is co-editor, with Meredith Blackwell, of Insect-Fungal Associations: Ecology and Evolution, published by Oxford University Press in 2005, and serves as an Editorial Board Member for Fungal Ecology.
A novel approach for biological control of insect pests could be the use of the endophytic entomopathogenic Beauveria bassiana isolate ATP-02. For the utilization of the endophyte as a commercial biocontrol agent, the fungus has to be mass-produced. B. bassiana was raised in shake flask cultures to produce high concentrations of total spores (TS), which include blastospores (BS) and submerged conidiospores (SCS). The highest concentration of 1.33×10(9) TS/mL and the highest yield of 5.32×10(10) TS/g sucrose was obtained in the TKI broth with 5% sugar beet molasses which consists of 50% sucrose as a carbon source. In spite of the lower sugar concentration (2.5%) the amount of TS could be increased up to 11-times in contrast to the cultivation with 5% sucrose. The scale-up to a 2 L stirred tank reactor was carried out at 25°C, 200-600 rpm and 1 vvm at pH 5.5. A TS yield of 5.2×10(10) TS/g sucrose corresponding to a SCS yield of 0.2×10(10) SCS/g sucrose was obtained after 216 h. With regards to the culture medium the cost of 10(12) TS amounts to 0.24 €. Plutella xylostella larvae, which were fed with oilseed rape leaves treated with spores from fermentation resulted in 77 ± 5% mortality. Moreover, spores from submerged cultivation were able to colonize oilseed rape leaves via leaf application. This is the first report of fermentation of an endophytic B. bassiana strain in a low-cost culture medium to very high yields of TS.
Under submerged growth in a defined medium (TKI broth), the entomopathogenic fungus, Beauveria bassiana, produced conidia; it produced only blastospores in complex media. Production of such "submerged" conidia depended on the nature of the carbon source and the presence of nitrate as a nitrogen source. Maximum yield of conidia (5 × 108 mL) was obtained when glucose was the carbon source and when the glucose to nitrate ratio was 5:1. Other carbon sources gave rise to both conidia and blastospores. Reducing the phosphate concentration resulted in the production of conidia which resembled "aerial" conidia in morphology and germination rates. The surfaces of "submerged" conidia were relatively smooth, but had a tendency to acquire the rough, warty, brittle surface characteristics of aerial conidia. Blastospores produced in defined media gave rise to conidia through microcycle conidiation without going through the vegetative phase of growth. In more complex media, blastospores did not undergo microcycle conidiation.
The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a widespread pest species of various cultivated plants. The pest status of FAW in cotton in the Cerrado region of Brazil has increased with the recent changes in cotton crop systems, such as double cropping and the use of cover or winter crops with non-tillage cropping systems. In this study we investigated the performance of FAW on three major crops cultivated in the Cerrado – soybean, corn, and cotton – and millet which has been integrated into the landscape as a cover crop. Further, the damage to various reproductive structures of cotton plants by FAW larvae was determined. Both studies were conducted under field conditions. Survival of FAW larvae caged on millet plants was higher than on other hosts. The FAW reared on millet also exhibited a net reproductive rate similar to that observed on corn, which was considered the best host for FAW, and the highest intrinsic rate of increase and lowest mean generation time compared to all other hosts. In cotton, the low availability of bolls during the plant’s blooming stage resulted in higher square feeding, whereas infestation during the plant’s boll stage resulted in higher loss of bolls and lower attack on squares. The number of adults produced in a generation was higher when plants were infested in the boll stage. The results indicated that FAW is a threat to cotton when bolls are available and can cause significant loss of reproductive structures. In addition, based on the FAW performance feeding on millet, this cover crop can be among the reasons of FAW increasing pest status in subsequent crops.
Growth and development of B. bassiana was followed in four liquid media: peptone, peptone-glucose, glucose and glucose-peptone-yeast extract. Six developmental stages were defined: (I) the unswollen conidium, (II) the swollen conidium, (III) emergence of the germ tube, (IV) elongation of the germ tube and formation of the first septum, (V) polar and bipolar elongation (growth) of the resulting mycelium and initiation of a blastospore and, (VI) seccession of that blastospore. Conidia of B. bassiana produced germ tubes in all liquid media. Blastospores were produced in all liquid media except glucose. In peptone-glucose, the yield of blastospores was four-fold higher than in glucose-peptone-yeast extract. However, biomass production was highest in peptone-glucose-yeast extract.
Spores of fungal pathogens of weeds and insects are unique in their ability to actively infect and kill their pest host.
While these capabilities are advantageous in terms of their use as a contact biological control agent, or biopesticide, they
also require special consideration during spore production. Directed approaches to medium optimization must consider not
only spore yield but also spore qualities such as desiccation tolerance, stability as a dry preparation, and biocontrol
efficacy. Nutritional conditions during culture growth and sporulation should direct the accumulation of appropriate endogenous
reserves so that newly formed spores possess these advantageous qualities. Studies with the bioherbicidal fungus Colletotrichum truncatum and with the bioinsecticidal fungus Paecilomyces fumosoroseus have demonstrated the impact of nutrition on spore ‘fitness’ for use as a biological control agent. The optimization strategy
used in these nutritional studies as well as a comparison of the results are presented.
A substantial number of mycoinsecticides and mycoacaricides have been developed worldwide since the 1960s. Here we present an updated, comprehensive list of these products. At least 12 species or subspecies (varieties) of fungi have been employed as active ingredients of mycoinsecticides and mycoacaricides for inundative and inoculative applications, although some are no longer in use. Products based on Beauveria bassiana (33.9%), Metarhizium anisopliae (33.9%), Isaria fumosorosea (5.8%), and B. brongniartii (4.1%) are the most common among the 171 products presented in this paper. Approximately 75% of all listed products are currently registered, undergoing registration or commercially available (in some cases without registration), whereas 15% are no longer available. We were unable to determine the status of the remaining 10%. Insects in the orders Hemiptera, Coleoptera, Lepidoptera, Thysanoptera, and Orthoptera comprise most of the targets, distributed among at least 48 families. A total of 28 products are claimed to control acarines (mites and ticks) in at least 4 families, although only three products (all based on Hirsutella thompsonii) were exclusively developed as acaricides. Eleven different technical grade active ingredients or formulation types have been identified, with technical concentrates (fungus-colonized substrates) (26.3%), wettable powders (20.5%) and oil dispersions (15.2%) being most common. Approximately 43% of all products were developed by South American companies and institutions. Currently, what may be the largest single microbial control program using fungi involves the use of M. anisopliae for control of spittlebugs (Cercopidae) in South American sugarcane and pastures.
Cryptococcus neoformans was first described as a human fungal pathogen more than a century ago. One aspect of the C. neoformans infectious life cycle that has been the subject of earnest debate is whether the spores are pathogenic. Despite much speculation,
no direct evidence has been presented to resolve this outstanding question. We present evidence that C. neoformans spores are pathogenic in a mouse intranasal inhalation model of infection. In addition, we provide mechanistic insights into
spore-host interactions. We found that C. neoformans spores were phagocytosed by alveolar macrophages via interactions between fungal β-(1,3)-glucan and the host receptors Dectin-1
and CD11b. Moreover, we discovered an important link between spore survival and macrophage activation state: intracellular
spores were susceptible to reactive oxygen-nitrogen species. We anticipate these results will serve as the basis for a model
to further investigate the pathogenic implications of infections caused by fungal spores.
Acyl-CoA synthetase (ACS) functions as a hub linking lipid metabolism with in cellular physiologies by producing active intermediate of catalyzes acyl-CoA. However, the biological roles of ACS are largely unknown in filamentous fungi. In this study, an ortholog of yeast Faa1, named BbFaa1, was functionally characterized in the filamentous entomopathogenic fungus Beauveria bassiana. BbFaa1 was associated with vesicular membrane, and its loss resulted in the impaired cytomembrane integrity. Notably, in ΔBbfaa1 mutant strain, the translocation of hydrophobins across cell membrane was significantly hampered, which resulted in the reduced hydrophobicity of aerial mycelia and conidia. In addition, loss of BbFaa1 significantly weakened fungal virulence. Our findings indicate that the metabolism of acyl-CoA synthetase Faa1 contributes to the cytomembrane functionality which cascades hydrophobin translocation and differentiation, thus affecting virulence of B. bassiana.
Blastospores of entomopathogenic fungi are yeast-like cells infective for arthropods, yet due to their thin-walled unicellular and hydrophilic properties, they are more susceptible than aerial conidia to abiotic stresses that may impair their efficacy and survival after field applications. We investigated the comparative susceptibility to heat and UV-B radiation of blastospores and aerial conidia of Metarhizium spp. (Metarhizium robertsii IP 146, Metarhizium anisopliae s.l. IP 363 and Metarhizium acridum ARSEF 324) and Beauveria bassiana s.l. (IP 361 and CG 307). Conidia and blastospores of each isolate were produced in solid and liquid Adámek modified medium, respectively, and then exposed to heat (45 ± 0.2 °C) in a range of 0 (control) to 360 min; the susceptibility of fungal propagules to heat exposures was assessed to express relative viability. Similarly, both propagules of each isolate were also exposed to a range of 0 (control) to 8.1 kJ m⁻² under artificial UV-B radiation. Our in vitro results show that fungal isolates, propagule types and exposure time or dose of the stressor source play critical roles in fungal survival challenged with UV-B and heat. Aerial conidia of ARSEF 324, IP 363, IP 146 and IP 361 exposed to heat survived significantly longer than their blastospores, except for blastospores of CG 307. Conidia and blastospores of IP 146 and IP 363 were equally tolerant to UV-B radiation, whereas the conidia of ARSEF 324, CG 307 and IP 361 exhibited greater tolerance than blastospores. Furthermore, ARSEF 324 exhibited the highest tolerance to both heat and UV-B stresses among all fungal isolates tested. We claim that blastospores of certain isolates may be promising candidates to control arthropod pests in tropical and subtropical regions where heat and UV-B are limiting environmental factors.
First we describe the different types of biocontrol used in greenhouses and present examples of each type. Next we summarize the history of greenhouse biocontrol, which started in 1926, showed a problematic period when synthetic chemical pesticides became available after 1945, and flourished again since the 1970s. After 1970, the number of natural enemies becoming available for commercial augmentative biocontrol in greenhouses grew very fast, as well as the industry producting these control agents. Biocontrol of the most important clusters of greenhouse pests is summarized, as well as the taxonomic groups of natural enemies that play a main role in greenhouses. More than 90% of natural enemy species used in greenhouses belong to the Arthropoda and less than 10%, many belonging to the Nematoda, are non-arthropods. This is followed by sections on finding and evaluation of potential biocontrol agents, and on mass production, storage, release and quality control of natural enemies. Since the 1970s, production of biocontrol agents has moved from a cottage industry to professional research and production facilities. Many efficient agents have been identified, quality control protocols, mass-production, shipment and release methods matured, and adequate guidance for farmers has been developed. Most natural enemy species (75%) are produced in low or medium numbers per week (hundreds to a hundred thousand), and are applied in situations where only low numbers are needed, such as private gardens, hospitals, banks, and shopping malls. The other 25% of the species are produced in numbers of 100,000 to up to millions per week and regularly released in many of the greenhouse crops. Microbial pesticides are predominantly used as corrective treatments in greenhouse crops where natural enemies are providing insufficient control. Europe is still the largest commercial market for arthropod greenhouse biocontrol agents, and North America is the largest market for microbial control agents. We then continue with a discussion on the pros and cons of use of polyphagous predators, and the use of semiochemicals. Finally, we summarize factors that indicate a positive future for greenhouse biocontrol, as well as developments frustrating its implementation.
Blastospores are yeast-like infective cells to arthropod pests and can be obtained shortly by liquid culture. However, fungal isolates exhibit striking variation in production, desiccation tolerance and virulence. In this study, we screened three isolates of Beauveria bassiana (Balsamo) Vuillemin and three of Cordyceps fumosorosea (Wize) (Hypocreales: Cordycipitaceae) for blastospore production and assessed desiccation tolerance by air drying. The virulence of these isolates were determined against Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype B, Spodoptera frugiperda (J.E. Smith) and Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae). The isolates ESALQ-1296 and ESALQ-3422 of C. fumosorosea and ESALQ-4958 of B. bassiana yielded blastospore concentrations above 1 × 109 ml−1, but only the two C. fumosorosea isolates retained viability rates above 75% after drying. Blastospores of both fungal species were more effective in killing the three insect species than aerial conidia. In summary, blastospores of C. fumosorosea ESALQ-1296 hold the best promise for broad-spectrum pest control in soybean.
Blastospores are yeast-like cells produced by entomopathogenic fungi that are infective to arthropods. The economical feasible production of blastospores of the insect killing fungus Metarhizium spp. must be optimized to increase yields. Moreover, stabilization process is imperative for blastospore formulation as a final product. In this sense, our goal was to increase blastospore production of two Metarhizium isolates (ESALQ1426 and ESALQ4676) in submerged liquid cultures. A modified Adamek's medium was supplemented with increased glucose concentrations and the fermentation time was accelerated by using a blastospore pre-culture as inoculum. Virulence of air-dried stable blastospores was compared with conidia toward larvae of the cattle tick, Rhipicephalus microplus. Our results revealed that blastospore production of Metarhizium is isolate- and species-dependent. Glucose-enriched cultures (140 g glucose/L) inoculated with pre-cultures improved yields with optimal growth conditions attained for Metarhizium robertsii ESALQ1426 that rendered as high as 5.9 × 10⁸ blastospores/mL within 2 d. Resultant air-dried blastospores of ESALQ1426 were firstly proved to infect and quickly kill cattle tick larvae with comparable efficiency to conidia. Altogether, we argue that both osmotic pressure, induced by high glucose titers, and isolate selection are critical to produce high yields of blastospores that hold promise to control cattle-tick larvae.
Ultraviolet radiation from sunlight is probably the most detrimental environmental factor affecting the viability of entomopathogenic fungi applied to solar-exposed sites (e.g., leaves) for pest control. Most entomopathogenic fungi are sensitive to UV radiation, but there is great inter- and intraspecies variability in susceptibility to UV. This variability may reflect natural adaptations of isolates to their different environmental conditions. Selecting strains with outstanding natural tolerance to UV is considered as an important step to identify promising biological control agents. However, reports on tolerance among the isolates used to date must be analyzed carefully due to considerable variations in the methods used to garner the data. The current review presents tables listing many studies in which different methods were applied to check natural and enhanced tolerance to UV stress of numerous entomopathogenic fungi, including several well-known isolates of these fungi. The assessment of UV tolerance is usually conducted with conidia using dose-response methods, wherein the UV dose is calculated simply by multiplying the total irradiance by the period (time) of exposure. Although irradiation from lamps seldom presents an environmentally realistic spectral distribution, laboratory tests circumvent the uncontrollable circumstances associated with field assays. Most attempts to increase field persistence of microbial agents have included formulating conidia with UV protectants; however, in many cases, field efficacy of formulated fungi is still not fully adequate for dependable pest control.
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 (>1×10(9)mL(-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 >13months 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.
Copyright © 2014. Published by Elsevier Inc.
Metarhizium anisopliae var. acridum (IMI 330189) can produce at least three spore types in vitro ; blastospores, submerged conidia, and aerial conidia, as defined by culturing conditions, sporogenesis, and spore morphology. This study compares morphological characteristics (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), and performance (germination rate and drying stability) among these three spore types. Submerged conidia and aerial conidia both possessed thick, double-layered cell walls, with hydrophobic regions on their surfaces. However, in contrast to aerial conidia, submerged conidia have: (1) a greater affinity for the lectin concanavalin-A; (2) more anionic net surface charge; and (3) a less distinct outer rodlet layer. Blastospores were longer and more variable in length than both submerged conidia and aerial conidia, and had thinner single-layered cell walls that lacked an outer rodlet layer. Also, blastospores had a greater affinity than either conidia type for the lectin, wheat germ agglutinin. Blastaspores lacked hydrophobic regions on their surface, and had a lower anionic net surface charge than submerged conidia. In culture, blastospores germinated the fastest followed by submerged conidia, and then aerial conidia. Survival of submerged conidia and aerial conidia were similar after drying on silica gel, and was greater than that for blastospores. We provide corroborating information for differentiating spore types previously based on method of production, sporogenesis, and appearance of spores. These physical characteristics may have practical application for predicting spore-performance characteristics relevant to production and efficacy of mycoinsecticides.
Determinations were made of the median lethal concentration (LC50) and median lethal time (LT50) of conidia and blastospores of B. bassiana against adult Nephotettix virescens (green leafhoppers). There was no significant difference between the LC50 values for blastospores produced in carbon- and nitrogen-limited cultures, but both types of blastospores were significantly less virulent than conidia. The LT50 values of conidia and of blastospores from nitrogen-limited cultures were significantly lower than the value for blastospores produced in carbon-limited culture. The LT50 values of conidia and blastospores increased during storage at 25 °C and were inversely related to spore viability.
At all but one concentration tested, nitrogen-limited blastospores ‘adhered’ to insects more strongly than did carbon-limited blastospores. Fewer spores germinated on insect wings than on agar medium and of the three spore types studied, maximal germination on insect wings was observed with nitrogen-limited blastospores.
The commercial use of entomopathogenic fungi and their products as mycoinsecticides necessitates their registration. Worldwide, several registration guidelines are available, however, most of them focus on similar or even the same safety issues. With respect to the two entomopathogenic fungi, Beauveria bassiana (Bals.-Criv.) Vuill. and Beauveria brongniartii (Sacc.) Petch, many commercial products have been developed, and numerous papers on different biological, environmental, toxicological and other safety aspects have been published during the past 30–40 years. The aim of the present review is to summarise these data. The following safety issues are presented: (1) identity of Beauveria spp.; (2) biological properties of Beauveria spp. (history, natural occurrence and geographical distribution, host range, mode of action, production of metabolites/toxins, effect of environmental factors); (3) analytical methods to determine and quantify residues; (4) fate and behaviour in the environment (mobility and persistence in air, water and soil); (5) effects on non-target organisms (non-target microorganisms, plants, soil organisms, aquatic organisms, predators, parasitoids, honey bees, earth worms and nontarget arthropods); (6) effects on vertebrates (fish, amphibia, reptiles and birds); and (7) effects on mammals and human health. Based on the present knowledge it is concluded that both Beauveria species are considered to be safe.
Beauveria bassiana in liquid culture can produce blastospores and occasionally submerged conidia. For use as a bioinsecticide, conidia have definite advantages. Numerous studies have investigated conidia production in liquid cultures using synthetic and industrial grade media supplemented with glucose. We have studied growth, development and sporulation in microcultures using growth media containing chitin monomers. For the production of submerged conidia growth media containing N-acetyl-d-glucosamine (GlcNAc) proved to be better than yeast extract-peptone-glucose (YPG), glucose plus ammonium salts (Glc+NH4Cl) or N-acetyl-d-galactosamine (GalNAc). Sixty-one percent of the spores in the GlcNAc medium were submerged conidia with the remainder being blastospores. The concentration of submerged conidia reached 8.0 105/ ml after two days in GlcNAc medium as compared to 8.9 105/ml in YPG medium. Therefore, in terms of percentage of submerged conidia produced, GlcNAc medium generated more submerged conidia in spite of its lower cell yields. Growth in a medium containing chitin, a polymer of GlcNAc, resulted in 86.3% of the spores as submerged conidia exceeding 106/ml after 48 h. Growth under phosphate limitation resulted in an increased percentage of submerged conidia for all media tested. Electron microscopy and spore protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that structural and compositional differences exist between the spore types.
Beauveria bassiana can produce three spore types; aerial conidia, submerged conidia and blastospores. We have examined the spore surface characteristics (hydrophobicity and cell-wall surface lectins), thermal inactivation and the virulence towards the migratory grasshopper, Melanoplus sanguinipes, of each of the three spore types. The hydrophobicities of the aerial and submerged conidia were quite similar. Blastospores were less hydrophobic than either of the two types of conidia. Hydrophobic interactions are thought to play a significant role in attachment of the spore to the host organism. However, the less hydrophobic blastospores were slightly more virulent (LT50 of 6.50 days) when compared to the aerial and submerged conidia (7.12 and 7.24 days), respectively. The lectin-binding characteristics of the aerial and submerged conidia were very similar but differed from that of blastospores. Growth of blastospores on a variety of carbohydrates did not affect their lectin-binding characteristics. Spore viability measurements showed that aerial and submerged conidia retained their viability for a longer period than blastospores. The similarity in hydrophobicity, stability, virulence and lectin-binding of aerial and submerged conidia make the latter an ideal candidate for mycoinsecticide production since they can be recovered after growth on inexpensive substrates.
Paecilomyces fumosoroseus was grown in submerged culture on complex and defined media. Growth on complex medium was followed by dry weight, absorbance and ATP measurements and all methods gave similar specific growth rates (0.240–0.290 h−1). In carbon-‘limited’ cultures, the organism produced blastospores rapidly during late exponential phase and continued to form them during stationary phase; some newly-formed spores germinated during the incubation period (152 h). Blastospore yield (number of spores cm−3 culture) in batch culture was not increased when the carbon source in the medium was varied to change specific growth rate of the organism (from 0.017 to 0.224 h−1), or when the concentration of (NH4)2SO4 in the medium was varied so that it was the first nutrient to become exhausted (nitrogen-‘limited’ cultures). However, there were appreciable differences in modal volumes and glycogen and nitrogen contents of blastospores produced in nitrogen-and carbon‘limited’ cultures.
Liquid media with differing carbon concentrations and carbon-to-nitrogen ratios were tested for production of desiccation tolerant blastospores of Paecilomyces fumosoroseus. While all media tested supported sporulation in submerged culture, high blastospore concentrations (5·8 × 108) spores ml−) were produced in media containing 80 g glucose l− and 13·2 g Casamino acids l− (MS medium) and a significantly higher percentage (79%) of these blastospores survived air drying. Media containing glucose concentrations greater than 20 g l − and Casamino acid concentrations between 13·2 and 40 g l− supported maximal production of desiccation tolerant blastospores. All 23 isolates of P. fumosoroseus grown in MS media produced high concentrations of desiccation tolerant blastospores. When stored at 4 °C, more than 60% of the lyophilized blastospores produced in MS medium were still viable after 7 months storage while less than 25% of the air-dried blastospores survived after 90 d storage. Standard whitefly bioassays were performed to compare air-dried blastospores of P. fumosoroseus ARSEF 4491 with solid substrate-produced conidia of Beauveria bassiana ARSEF 252. Air-dried blastospores of P. fumosoroseus gave LD50s of 60 and 113 blastospores mm− for the silverleaf whitefly (Bemisia argentifolii) in two separate bioassays with potency ratios (LD50B. bassiana/LD50P. fumosoroseus)of 3·9 and 3·8, respectively. These results have demonstrated that high concentrations of blastospores of P. fumosoroseus can be rapidly produced in liquid culture, remain viable following drying, and infect and kill silverleaf whitefly.
The entomopathogenic fungus Beauveria bassiana produces aerial conidia, submerged conidia or blastospores depending on the culture conditions. Rodlets are observed on aerial and submerged conidia but not on blastospores. Cell wall rodlets were removed by sonication and the proteins dissolved with formic acid. A single major 9·7 kDa protein was found in rodlets of aerial and submerged conidia and showed N terminal sequence similarities to the hydrophobin class of fungal proteins. Hydrophobin could not be recovered from blastospores. Oxidation of the hydrophobin with performic acid produced a protein of higher molecular weight (14·0 kDa). The rodlet layer could not be removed from intact conidia by boiling in sodium dodecyl sulphate (SDS) but could be removed with formic acid. Formic acid treated conidia retained the ability to bind to insect cuticles and retained hydrophobicity as indicated by a phase exclusion assay.
Transgenic maize, Zea mays L., event TC1507 produces the Cry1F protein to provide protection from feeding by several important lepidopteran pests, including Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae). Reports of reduced field performance against this species in Puerto Rico were investigated, and laboratory bioassays showed that S. frugiperda collected from the affected area exhibited lower sensitivity to the Cry1F protein compared with typical colonies from other regions. The resistance was shown to be autosomally inherited and highly recessive. The Puerto Rico colony was shown to be moderately less sensitive than susceptible laboratory strains to Cry1Ab and Cry1Ac, but the differences in sensitivity were dramatically smaller than for Cry1F. Potential contributory factors to the emergence of resistance to Cry1F in Puerto Rico populations of S. frugiperda include the tropical island geography, unusually large population sizes in 2006, and drought conditions reducing the availability of alternative hosts. In response to this resistance incident, the technology providers have stopped commercial sales of TC1507 maize in Puerto Rico pending potential reversion to susceptibility.
SUMMARY The ability of Aspergillus niger conidia to produce conidiophores after ger- mination in shaken culture at 30" was stimulated by the inclusion of glutamate in the medium. Incubation of the conidia at 35" to 41" increased swelling of the conidia and also the proportion which produced conidiophores. Although conidio phore initiation was stimulated at temperatures between 35" and 41 ", maturation was poor and optimum conidiation was obtained by incubation at these temper- atures followed by 30". Conidiophore formation from conidia required a prior period of spore metabolism and at temperatures between 30" and 41 " did not occur until several hours after germination. Direct conidiophore production from conidia in the complete absence of vegetative growth was achieved by incubation of the conidia at 44" (which allows only swelling) for a prolonged period (48 h.) followed by 30". Although vegetative growth was absent the conidiophores were similar to, but smaller than, normal subaerial conidiophores and viable conidia were produced. These conidia differed from subaerial spores in lacking the dark pigmented spore coat.
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