Community composition of the entomopathogenic fungal genus Metarhizium in soils of tropical and temperate conventional and organic strawberry fields.

Studies on community composition and population structure of entomopathogenic fungi are imperative to link ecosystem functions to conservation biological control. We studied the diversity and abundance of Metarhizium spp. from soil of conventionally and organically farmed strawberry crops and from the adjacent field margins in two different climatic zones: Brazil (tropical) and Denmark (temperate), using the same isolating methods. In Brazilian strawberry soil, Metarhizium robertsii (n = 129 isolates) was the most abundant species, followed by M. humberi (n = 16); M. anisopliae (n = 6); one new taxonomically unassigned lineage Metarhizium sp. indet. 5 (n = 4); M. pingshaense (n = 1) and M. brunneum (n = 1). In Denmark, species composition was very different, with M. brunneum (n = 33) being isolated most commonly, followed by M. flavoviride (n = 6) and M. pemphigi (n = 5), described for the first time in Denmark. In total, 17 haplotypes were determined based on MzFG543igs sequences, four representing Danish isolates and 13 representing Brazilian isolates. No overall difference between the two climatic regimes was detected regarding the abundance of Metarhizium spp. in the soil in strawberry fields and the field margins. However, we found a higher Shannon's diversity index in organically managed soils, confirming a more diverse Metarhizium community than in soils of conventionally managed agroecosystems in both countries. These findings contribute to the knowledge of the indigenous diversity of Metarhizium in agricultural field margins with the potential to contribute to pest regulation in strawberry cropping systems.

Characterization of Brazilian Cordyceps fumosorosea isolates: Conidial production, tolerance to ultraviolet-B radiation, and elevated temperature.

Cordyceps fumosorosea is an entomopathogenic fungus with a global distribution and is used for the biological control of agricultural pests. High conidial productivity and tolerance to abiotic stresses such as elevated temperature and ultraviolet radiation (UV-B) are desired characteristics in candidate isolates for commercial products. Our goal in this study was to characterize promising isolates of C. fumosorosea from five Brazilian biomes regarding conidial production, tolerance to UV-B, and elevated temperature (45°). Seventy-two isolates out of 172 were chosen visually, based on growth and sporulation in culture medium, and grown on parboiled rice. Next, fourteen isolates were selected, based on productivity on rice and origin of isolation, for production in polypropylene bags and submitted to UV-B for 2, 4, 6, and 8 h or to 45 °C for 30, 60, and 90 min. High variations in conidial production were observed among isolates, and a positive correlation was observed between UV-B and heat tolerance. The isolates ESALQ4556 and ESALQ4778 showed the highest yields of conidial production in polypropylene bags (3.51 × 109 conidia/g dry rice), while ESALQ1296, an isolate recovered from insects, was the most tolerant to UV-B and 45 °C. Exposure to radiation for more than 4 h and placed directly at 45 °C for more than 30 min significantly reduced conidial germination for all C. fumosorosea isolates. These results contribute to a better understanding of the tolerance to abiotic factors of Brazilian isolates of C. fumosorosea.

Development of novel spray-dried and air-dried formulations of Metarhizium robertsii blastospores and their virulence against Dalbulus maidis.

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. 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.

Current status and perspectives of fungal entomopathogens used for microbial control of arthropod pests in Brazil.

Entomopathogenic fungi play a central role in Brazil's biopesticide market. Approximately 50% of registered microbial biopesticides comprise mycoinsecticides and/or mycoacaricides consisting of hypocrealean fungi, with most based on Metarhizium anisopliae sensu stricto (s. str.) and Beauveria bassiana s. str. These fungi are mainly used to control spittlebugs in sugarcane fields and whiteflies in row crops, respectively, with annual applications surpassing three million hectares. Research also emphasizes the potential of fungal entomopathogens to manage arthropod vectors of human diseases. Most registered fungal formulations comprise wettable powders or technical (non-formulated) products, with relatively few new developments in formulation technology. Despite the large area treated with mycoinsecticides (i.e., approx. 2 million ha of sugarcane treated with M. anisopliae and 1.5 million ha of soybean treated with B. bassiana), their market share remains small compared with the chemical insecticide market. Nevertheless, several major agricultural companies are investing in fungus-based products with the aim at achieving more sustainable IPM programs for major pests in both organic and conventional crops. Government and private research groups are pursuing innovative technologies for mass production, formulation, product stability and quality control, which will support cost-effective commercial mycoinsecticides. Here, we summarize the status of mycoinsecticides currently available in Brazil and discuss future prospects.

Metarhizium humberi sp. nov. (Hypocreales: Clavicipitaceae), a new member of the PARB clade in the Metarhizium anisopliae complex from Latin America.

A new species, Metarhizium humberi, from the M. anisopliae complex and sister lineage of the M. anisopliae s.str. in the PARB clade, including M. pingshaense, M. anisopliae, M. robertsii and M. brunneum, is described based on phylogenetic analyses [translation elongation factor 1-alpha (5'TEF and 3'TEF), RNA polymerase II largest subunit (RPB1a), RNA polymerase II second largest subunit (RPB2a) and ß-tubulin (BTUB)]. Metarhizium humberi was first collected in 2001 in the Central Brazilian state of Goiás, later found to be a common fungus in soils in Brazil, and since then has also been isolated from coleopteran, hemipteran and lepidopteran insects in Brazil and Mexico. This new species, named in honor of Richard A. Humber, a well-known insect pathologist and taxonomist of entomopathogenic fungi, is characterized by a high insecticidal activity against different developmental stages of arthropod pests with importance in agriculture and vectors of diseases to human and animals.

Increased tolerance of Beauveria bassiana and Metarhizium anisopliae conidia to high temperature provided by oil-based formulations.

The influence of the temperature of aqueous conidial sprays on conidial viability and virulence against Diatraea saccharalis was evaluated for pure conidia, rice + fungus (technical concentrates) and oil-based formulations of Beauveria bassiana s.s. and Metarhizium anisopliae s.s. under laboratory conditions. The fungal preparations were suspended in water and maintained at 26 °C, 36 °C and 46 °C for one, four and six hours. Conidial viability was determined by plating aliquots of each suspension onto PDA medium followed by incubation for 20-22 h and observing for viable conidia (germ tubes longer than diameter of conidia). Fungal virulence was determined by spraying suspensions onto third-instar larvae of D. saccharalis. In general, germination and virulence, particularly for unformulated conidia, were negatively affected by increases in water temperature and exposure time in suspension. However, the decrease in conidial viability in the oil-in-water emulsion was less than 7% for both species after 6 h of exposure at 36 °C, in contrast to reductions of 7-21% and 28-60% for the oil-free suspensions of B. bassiana and M. anisopliae, respectively. For the sprays of conidia in an oil-in-water emulsion previously exposed to elevated water temperatures for longer periods, the levels of insect mortality were higher than those of pure conidia or technical concentrates under identical conditions. Our results indicate that emulsifiable oil-based formulations can protect the conidia of both species of fungi from the adverse effects of high water temperatures before spraying in the field.

Exploring virulence of new and less studied species of Metarhizium spp. from Brazil for two-spotted spider mite control.

The two-spotted spider mite Tetranychus urticae is an important pest of strawberry crops in Brazil and many other countries. Focus for biocontrol studies involving entomopathogenic fungi has been on three species from the genus Metarhizium: M. anisopliae sensu stricto (s.s.), M. brunneum and M. robertsii. Also, the species Beauveria bassiana has been studied for spider mite control and one isolate (ESALQPL63) is commercially available in Brazil. New and undescribed Metarhizium species have been found recently in Brazil and provide a pool of isolates with potential for biocontrol in Brazil and probably also elsewhere. The mortality of adult females of T. urticae when exposed to four new Brazilian species of Metarhizium was compared to the mortality when exposed to M. anisopliae s.s., M. brunneum, M. pingshaense, M. robertsii and Beauveria bassiana ESALQPL63. Fungal suspensions were sprayed onto mites at 107 conidia/mL with 0.05% Tween 80 in laboratory bio-assays. We measured total mortality and percentage sporulating cadavers 10 days after exposure and calculated median lethal time (LT50). The lowest LT50 (4.0 ± 0.17) was observed for mites treated with Metarhizium sp. Indet. 1 (ESALQ1638), which also performed well with respect to mortality after 10 days and capacity to sporulate from cadavers. Among the other little studied species tested, M. pingshaense (ESALQ3069 and ESALQ3222) and Metarhizium Indet. 2 (ESALQ1476) performed well and were comparable to B. bassiana (ESALQPL63). The new Metarhizium isolates and species thus showed potential for biological control.

Impacts of Nosema sp. (Microsporidia: Nosematidae) on the sugarcane borer, Diatraea saccharalis (Lepidoptera: Crambidae).

In Brazil, the sugarcane borer, Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae), is controlled with massive releases of the hymenopteran parasitoid Cotesia flavipes Cam. (Hymenoptera: Braconidae); over 3 million hectares of sugarcane are treated annually with 18 billion parasitoids. In order to meet this demand, parasitoids are produced in D. saccharalis under laboratory conditions where a Nosema sp. is reported to be an important problem in mass rearing of the host. The goals for this work were to study the pathogenicity of the Nosema sp. and the progression of the disease in the host under laboratory conditions. The average median lethal time (LT50) of Nosema sp. in first instar D. saccharalis varied from 9 ± 0.3 to 42 ± 2.3 days at concentration of 5 × 10(5)-0.5 spores/mm(3) artificial diet (10(7)-10 spores/µl). For third instar, the average of LT50 ranged from 32 ± 0.7 to 37 ± 0.7 days at concentration of 5 × 10(5)-5 × 10(2) spores/mm(3) artificial diet (10(7)-10(4) spores/µl in saline). The concentration necessary to cause 50% mortality (LC50) of first instar larvae was 5.6 (0.9-17.6) spores/µl and the estimated LC50 for third instar larvae was 1,200 (200-4700) spores/µl. The impacts of Nosema sp. on D. saccharalis were analyzed for first instar larvae fed 0.5 spores/mm(3) artificial diet. Duration and viability of the larval and pupal stages, adult longevity, pupal weight and fertility life table were measured for offspring of mating pairs composed of infected females and uninfected males or infected males and uninfected females and compared to offspring of uninfected pairs. Nosema sp. infection resulted in adverse effects on all biological parameters measured except for the duration of the larval and pupal stages and the weight of the male pupae, which did not differ statistically between infected and uninfected groups. The intrinsic rates of growth (rm) were greater for uninfected pairs compared to pairs with either male or female infected. The growth rate of individual larvae produced by uninfected adults was 48.2% faster than of larval offspring of infected females and it was negative (-0.003) when males were infected. Our study confirms the negative impact of the Nosema sp. in mass rearing of D. saccharalis for parasitoid production but shows potential for use as a microbial control agent of the sugarcane borer.

Two-spotted spider mite and its natural enemies on strawberry grown as protected and unprotected crops in Norway and Brazil.

Cultivation of strawberry in plastic tunnels has increased considerably in Norway and in southeastern Brazil, mainly in an attempt to protect the crop from unsuitable climatic factors and some diseases as well as to allow growers to expand the traditional production season. It has been hypothesized that cultivation under tunnels could increase the incidence of one of its major pests in many countries where strawberry is cultivated, including Norway and Brazil, the two spotted spider mite, Tetranychus urticae. The objective of this study was to evaluate the effect of the use of tunnels on the incidence of T. urticae and on its natural enemies on strawberry in two ecologically contrasting regions, Norway (temperate) and southeastern Brazil (subtropical). In both countries, peak densities of T. urticae in tunnels and in the open fields were lower than economic thresholds reported in the literature. Factors determining that systematically seem to be the prevailing relatively low temperature in Norway and high relative humidity in both countries. The levels of occurrence in Norway and Brazil in 2010 were so low that regardless of any potential effect of the use of tunnel, no major differences were observed between the two cropping systems in relation to T. urticae densities. In 2009 in Norway and in 2011 in Brazil, increase in T. urticae population seemed to have been restrained mainly by rainfall in the open field and by predatory mites in the tunnels. Phytoseiids were the most numerous predatory mite group of natural occurrence on strawberry, and the prevalence was higher in Brazil, where the most abundant species on strawberry leaves were Neoseiulus anonymus and Phytoseiulus macropilis. In Norway, the most abundant naturally occurring phytoseiids on strawberry leaves were Typhlodromus (Anthoseius) rhenanus and Typhlodromus (Typhlodromus) pyri. Predatory mites were very rare in the litter samples collected in Norway. Infection rate of the pest by the fungus Neozygites floridana (Neozygitaceae) was low. The results of this work suggest that in Norway the use of tunnels might not affect the population densities of T. urticae on strawberry in years of lower temperatures. When temperature is not a limiting factor for the development of T. urticae in that country (apparently always the case in southern Brazil), strawberry cultivation in the tunnels may allow T. urticae to reach higher population levels than in open fields (because of the provided protection from the direct impact of rainfall), but natural enemies may prevent higher levels from being reached.

Confirmation of Neozygites floridana azygospore formation in two-spotted spider mite (Tetranychus urticae) in strains from tropical and temperate regions.

Neozygites floridana is an obligate fungal pathogen of mites in the family Tetranychidae and is an important natural enemy of the two-spotted spider mite (Tetranychus urticae). Until now, information about the formation of azygospores remained to be fully confirmed. In this study, we document the formation of azygospores by a Brazilian N. floridana strain and the formation of azygospores and zygospores by a Norwegian N. floridana strain, both in the host T. urticae. Evidence of both zygosporogenesis and azygosporogenesis was also found in the same individual in the Norwegian stains. Further we report the presence of immature azygospores with 1-3 nuclei for the Norwegian strains, immature resting spores (probably azygospores) with 1-8 nuclei for the Brazilian strain, and mature resting spores with 2 nuclei for both the Norwegian and the Brazilian strains (azygo- or zygospores). Our observations suggest that the immature resting spore (prespore) of both strains begins in a multinucleate condition but that the nuclear number is reduced during maturation until mature resting spore is binucleate regardless of its origin as a zygospore or azygospore.

The effects of photoperiod and light intensity on the sporulation of Brazilian and Norwegian isolates of Neozygites floridana.

The objective of this study was to determine the effects of light intensity and duration (photoperiod) on the sporulation (discharge of primary conidia) and conidia germination (from non-infective primary conidia to infective capilliconidia) of Neozygites floridana isolates from Tetranychus urticae originating from Norway and Brazil. Two light intensities (40 and 208 µmolm(-2)s(-1)), three photoperiods (24 h of continuous light (24 h D), 12 h of darkness followed by 12 h of light (12 h D: 12 h L) and 24 h of continuous darkness (24 h D)) and two temperatures (18°C and 23°C) were tested. The fungus produced similar amounts of primary conidia and capilliconidia at 12 h D:12 h and 24 h D, indicating that the fungus discharges almost all of its conidia during the first 12 h of darkness. Light had less of an effect on the production of primary conidia than on capilliconidia formation. At 24 h L, capilliconidia formation was significantly lower for all tested light intensities, temperatures and isolates compared to 12 h D:12 h L and 24 h D. At both light intensities, 24 h L resulted in a significantly lower capilliconidia formation for the Norwegian isolate compared to the Brazilian isolate. Our data suggest that, even though 24 h L reduced sporulation, some capilliconidia formation may occur at the low light intensities found on the underside of strawberry leaves during parts of the day as well as the top of a non-shaded strawberry leaf during the dim evening and morning hours in the tropics and during the dim, long summer days in temperate regions.

Blastospores from Metarhizium anisopliae and Metarhizium rileyi Are Not Always as Virulent as Conidia Are towards Spodoptera frugiperda Caterpillars and Use Different Infection Mechanisms.

Infective conidia from entomopathogenic fungi are widely used to control insect pests. Many entomopathogenic fungi also produce yeast-like cells called blastospores under specific liquid culture conditions that can directly infect insects. However, little is known about the biological and genetic factors that allow blastospores to infect insects and make them potentially effective for biological control in the field. Here, we show that while the generalist Metarhizium anisopliae produces a higher number of and smaller blastospores, the Lepidoptera specialist M. rileyi produces fewer propagules with a higher cell volume under high-osmolarity conditions. We compared the virulence of blastospores and conidia of these two Metarhizium species towards the economically important caterpillar pest Spodoptera frugiperda. Conidia and blastospores from M. anisopliae were equally infectious, but acted slower, and killed fewer insects than M. rileyi conidia and blastospores did, where M. rielyi conidia had the highest virulence. Using comparative transcriptomics during propagule penetration of insect cuticles, we show that M. rileyi blastospores express more virulence-related genes towards S. frugiperda than do M. anisopliae blastospores. In contrast, conidia of both fungi express more virulence-related oxidative stress factors than blastospores. Our results highlight that blastospores use a different virulence mechanism than conidia use, which may be explored in new biological control strategies.

Natural occurrence of Zoophthora radicans (Entomophthorales: Entomophthoraceae) on Thaumastocoris peregrinus (Heteroptera: Thaumastocoridae), an invasive pest recently found in Brazil.

Occurrence of Zoophthora radicans infecting nymphs and adults of Thaumastocoris peregrinus Carpintero and Dellape, 2006 is reported in Brazil. This is a new record of host for this fungal species and the first fungal pathogen associated with this pest worldwide. Infection of Z. radicans on T. peregrinus populations on commercial Eucalyptus plantation (Eucalyptus spp.) reached up to 100%, and low insect densities were associated with high levels of fungal infection in three out of seven plots. This pathogen seems to be virulent against T. peregrinus and may play an important role in population regulations of this invasive pest through naturally induced epizootics.

Laboratory Risk Assessment of Three Entomopathogenic Fungi Used for Pest Control toward Social Bee Pollinators.

The use of fungal-based biopesticides to reduce pest damage and protect crop quality is often considered a low-risk control strategy. Nevertheless, risk assessment of mycopesticides is still needed since pests and beneficial insects, such as pollinators, co-exist in the same agroecosystem where mass use of this strategy occurs. In this context, we evaluated the effect of five concentrations of three commercial entomopathogenic fungi, Beauveria bassiana, Metarhizium anisopliae, and Cordyceps fumosorosea, by direct contact and ingestion, on the tropical stingless bees Scaptotrigona depilis and Tetragonisca angustula, temperate bee species, the honey bee Apis mellifera, and the bumble bee Bombus terrestris, at the individual level. Furthermore, we studied the potential of two infection routes, either by direct contact or ingestion. In general, all three fungi caused considerable mortalities in the four bee species, which differed in their response to the different fungal species. Scaptotrigona depilis and B. terrestris were more susceptible to B. bassiana than the other fungi when exposed topically, and B. terrestris and A. mellifera were more susceptible to M. anisopliae when exposed orally. Interestingly, increased positive concentration responses were not observed for all fungal species and application methods. For example, B. terrestris mortalities were similar at the lowest and highest fungal concentrations for both exposure methods. This study demonstrates that under laboratory conditions, the three fungal species can potentially reduce the survival of social bees at the individual level. However, further colony and field studies are needed to elucidate the susceptibility of these fungi towards social bees to fully assess the ecological risks.

Transcriptional Responses of Beauveria bassiana Blastospores Cultured Under Varying Glucose Concentrations.

Culturing the entomopathogenic fungus, Beauveria bassiana, under high glucose concentrations coupled with high aeration results in a fungal developmental shift from hyphal growth to mostly blastospores (yeast-like cells). The underlying molecular mechanisms involved in this shift remain elusive. A systematic transcriptome analysis of the differential gene expression was preformed to uncover the fungal transcriptomic response to osmotic and oxidative stresses associated with the resulting high blastospore yield. Differential gene expression was compared under moderate (10% w/v) and high (20% w/v) glucose concentrations daily for three days. The RNAseq-based transcriptomic results depicted a higher proportion of downregulated genes when the fungus was grown under 20% glucose than 10%. Additional experiments explored a broader glucose range (4, 8, 12, 16, 20% w/v) with phenotype assessment and qRT-PCR transcript abundance measurements of selected genes. Antioxidant, calcium transport, conidiation, and osmosensor-related genes were highly upregulated in higher glucose titers (16-20%) compared to growth in lower glucose (4-6%) concentrations. The class 1 hydrophobin gene (Hyd1) was highly expressed throughout the culturing. Hyd1 is known to be involved in spore coat rodlet layer assembly, and indicates that blastospores or another cell type containing hydrophobin 1 is expressed in the haemocoel during the infection process. Furthermore, we found implications of the HOG signaling pathway with upregulation of homologous genes Ssk2 and Hog1 for all fermentation time points under hyperosmotic medium (20% glucose). These findings expand our knowledge of the molecular mechanisms behind blastospore development and may help facilitate large-scale industrial production of B. bassiana blastospores for pest control applications.

UV-B Radiation Tolerance and Temperature-Dependent Activity Within the Entomopathogenic Fungal Genus Metarhizium in Brazil.

Metarhizium comprises a phylogenetically diverse genus of entomopathogenic fungi. In Brazil, Metarhizium anisopliae s.str. subclade Mani 2 is predominantly isolated from insects, while M. robertsii and M. brunneum mostly occur in the soil environment. Solar radiation and high temperatures are important abiotic factors that can be detrimental to fungal propagules. We hypothesized that among 12 Brazilian isolates of Metarhizium spp., M. anisopliae Mani 2 (n = 6), being adapted to abiotic conditions of the phylloplane, is more tolerant to UV light and high temperatures than M. robertsii (n = 3) and M. brunneum (n = 3). Inoculum of each isolate was exposed to UV-B for up to 8 h and viability evaluated 48 h later. After 8 h under UV-B, most of the isolates had germination rates below 5%. Discs of mycelia were incubated at different temperatures, and diameter of colonies were recorded for 12 days. Mycelia of M. robertsii isolates grew faster at 33 °C, while M. anisopliae and M. brunneum grew most at 25 °C. Dry conidia were incubated at 20, 25 or 40 °C for 12 days, and then viabilities were examined. At 40 °C, conidia of five M. anisopliae isolates were the most tolerant. In the three experiments, considerable intra- and inter-specific variability was detected. The results indicate that conclusions about tolerance to these abiotic factors should be made only at the isolate level.

Fungal isolate and crop cultivar influence the beneficial effects of root inoculation with entomopathogenic fungi in strawberry.

BACKGROUND: Root inoculations of crop plants with beneficial fungi constitute a promising strategy for growth promotion and control of above-ground pests and diseases. Here, strawberry roots (cultivar 'Albion' and 'Pircinque') were inoculated with 25 different Brazilian entomopathogenic fungal isolates of three genera and the effects on Tetranychus urticae oviposition and plant growth were evaluated in greenhouse experiments. RESULTS: Reductions in the number of T. urticae eggs compared to control treatments were observed on both cultivars inoculated with almost all isolates. For the cultivar 'Albion', Metarhizium anisopliae (ESALQ 1604, ESALQ 1669), M. robertsii (ESALQ 1622, ESALQ 1635), Metarhizium sp. Indet. (ESALQ 1684) and Beauveria bassiana (ESALQ 3323) increased dry weight of roots and leaves, and fruit yield, while M. robertsii (ESALQ 1634), Metarhizium sp. Indet. (ESALQ 1637) and (ESALQ 1636) enhanced fruit yield and dry weight of leaves, respectively. For the cultivar 'Pircinque', M. anisopliae (ESALQ 1669) was the only isolate observed to increase dry weight of roots. CONCLUSION: The results suggest that inoculation of strawberry roots with entomopathogenic fungi may be an innovative strategy for pest management above ground. Furthermore, these inoculations may also stimulate plant growth and strawberry production, but the effects depend on fungal strains and crop cultivar. © 2019 Society of Chemical Industry.

Host range of Neozygites floridana isolates (zygomycetes: entomophthorales) to spider mites.

Neozygites floridana (Weiser & Muma) (Zygomycetes: Entomophthorales) has been reported infecting naturally at least 18 species of tetranychids worldwide. However, the host range of N. floridana is unknown. Epizootics caused by this pathogen to tetranychid populations indicate that N. floridana has the potential to be used as a biological control agent. However, the virulence and specificity of species and strains of Neozygites need to be assessed in the laboratory to reveal its potential as a biological control agent. N. floridana isolates are currently been investigated in Brazil as biological control agents against the tomato red mite, Tetranychus evansi Baker & Pritchard, and the two-spotted spider mite, Tetranychus urticae Koch. The pathogenicity of five strains of N. floridana obtained from T. urticae, T. evansi and T. ludeni Zacher was assessed against populations of Mononychellus tanajoa (Bondar), Schizotetranychus sacharum Flechtmann & Baker, Tetranychus abacae Baker & Pritchard and Tetranychus armipenis Flechtmann & Baker, in addition to the species from which the fungus was obtained. Mummified mites were placed on leaf discs of the host plant of each tetranychid to promote fungal sporulation, and after 24h the mites were transferred to the leaf discs. Contamination, infection and mummification were evaluated daily for seven days after confinement. Each isolate was pathogenic to three or four out of the six spider mite species tested. However, except for isolate ESALQ1421, all isolates caused higher levels of infection and significant mummification only to the tetranychid species from which they were collected. None of the isolates was pathogenic to S. sacharum and only one isolate infected T. abacae. Alternative hosts may be important for N. floridana survival in tropical regions where resting spores are rarely found.

Role of entomopathogenic fungi in the control of Tetranychus evansi and Tetranychus urticae (Acari: Tetranychidae), pests of horticultural crops.

The spider mites Tetranychus urticae Koch and Tetranychus evansi Baker and Pritchard are important pests of horticultural crops. They are infected by entomopathogenic fungi naturally or experimentally. Fungal pathogens known to cause high infection in spider mite populations belong to the order Entomophthorales and include Neozygites spp. Studies are being carried out to develop some of these fungi as mycoacaricides, as stand-alone control measures in an inundative strategy to replace the synthetic acaricides currently in use or as a component of integrated mite management. Although emphasis has been put on inundative releases, entomopathogenic fungi can also be used in classical, conservation and augmentative biological control. Permanent establishment of an exotic agent in a new area of introduction may be possible in the case of spider mites. Conservation biological control can be achieved by identifying strategies to promote any natural enemies already present within crop ecosystems, based on a thorough understanding of their biology, ecology and behaviour. Further research should focus on development of efficient mass production systems, formulation, and delivery systems of fungal pathogens.

Prediction of Sporulation and Germination by the Spider Mite Pathogenic Fungus Neozygites floridana (Neozygitomycetes: Neozygitales: Neozygitaceae) Based on Temperature, Humidity and Time.

Neozygites floridana is a pathogenic fungus and natural enemy of the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), which is an important polyphagous plant pest. The aim of this study was to reveal and predict what combination of temperature, relative humidity (RH), and time that enables and promotes primary conidia production and capilliconidia formation in N. floridana (Brazilian isolate ESALQ 1420), in both a detached leaf assay mimicking climatic conditions in the leaf boundary layer and in a semi-field experiment. In the detached leaf assay, a significant number of conidia were produced at 90% RH but the highest total number of primary conidia and proportion of capilliconidia was found at 95 and 100% RH at 25 °C. Positive temperature and RH effects were observed and conidia production was highest in the 8 to 12 h interval. The semi-field experiment showed that for a >90% probability of N. floridana sporulation, a minimum of 6 h with RH >90% and 10 h with temperatures >21 °C, or 6 h with temperatures >21 °C and 15 h with RH >90% was needed. Our study identified suitable conditions for primary- and capilliconidia production in this Brazilian N. floridana isolate. This information provides an important base for building models of a Decision Support System (DSS) where this natural enemy may be used as a tool in Integrated Pest Management (IPM) and a base for developing in vivo production systems of N. floridana.