First survey on ecological host range of aphid pathogenic fungi (Phylum Entomophthoromycota) in Tunisia

Summary. The natural occurrence of fungal pathogens of aphids and their ecological host range was investigated in Tunisia from 2009 to 2012. The survey focused on aphid infesting different crops and weeds and included 10 different aphid species. Samples were collected from eight agricultural crops sites belonging to three different bioclimatic zones. Four pathogens from the phylum Entomophthoromycota were found to occur naturally in Tunisian ecosystems: Pandora neoaphidis (Entomophthorales: Entomophthoraceae), Entomophthora planchoniana (Entomophthorales: Entomophthoraceae), Conidiobolus obscurus (Entomophthorales: Ancylistaceae) and Neozygites fresenii (Neozygitales: Neozygitaceae). The occurrence of entomophthoralean fungi depended on the sampling area, the bioclimatic zone, and aphid species. P. neoaphidis and E. planchoniana were the predominant pathogens infecting a wide range of aphid species whereas C. obscurus and N. fresenii were sporadically present on a limited number of aphid species. This study is the first survey on ecological host range of entomophthoralean fungi in Tunisia, and the first documentation of C. obscurus and N. fresenii to occur in Tunisia and Maghreb Region.     

Exploring virulence of new and less studied species of <Emphasis Type="Italic">Metarhizium</Emphasis> 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.     

Secretome of fungus-infected aphids documents high pathogen activity and weak host response

The discovery of novel secretome proteins can add to our understanding of host-pathogen interactions. Here we report a rich diversity of secreted proteins from the interaction between grain aphids (host, insect order Hemiptera) and fungi of the order Entomophthorales (insect pathogens). The proteins were identified using a unique method unbiased by known sequences or functions to screen a cDNA library constructed directly from field-sampled material. We show for the first time that fungi from the genera Pandora and Entomophthora are armed with a battery of hydrolytic enzymes for penetrating the host cuticle. This enables both access to the hemolymph and exit for sporulation. Further, they secrete enzymes, most notably a number of lipases, for digestion of easily accessible high-energy compounds in the hemolymph. In contrast, we identified only few host genes potentially involved in the interaction, indicating that aphids respond only weakly to the pathogens. These results support recent findings that aphids have a reduced immune repertoire.     

Intraspecific variation and host specificity of Entomophthora muscae sensu stricto isolates revealed by random amplified polymorphic DNA,universal primed PCR,PCR-restriction fragment length polymorphism,and conidial morphology

The intraspecific variations of Entomophthora muscae s. str. associated with particular host species, Musca domestica and Delia radicum, sampled from different localities and different years in Denmark and the variation of E. muscae s. str. originating from different host taxa were investigated. The isolates were compared both by primary spore morphology and by three molecular methods: random amplified polymorphic DNA, universal primed PCR, and PCR-restriction fragment length polymorphism. Analyses of the different molecular data showed the same overall picture and separated E. muscae s. str. into two main groups with all the M. domestica isolates in one group and isolates from D. radicum, Coenosia tigrina, and Pegoplata infirma in the second group. E. muscae s. str. isolates from M. domestica also differ significantly from the rest of the E. muscae s. str. isolates with regard to the morphology of the primary conidia, which were bigger and contained significantly more nuclei per conidium. Several different E. muscae s. str. genotypes were documented and each type was restricted to a single host species, indicating a very high degree of host specificity at or below the level of the subfamily.     

Dispersal of Beauveria bassiana by the activity of nettle insects

Recent studies have shown that the entomopathogenic fungus Beauveria bassiana occurs naturally on the phylloplanes of several plants, including nettles. Insects could, by their activity, be contributing to this inoculum by dispersing it from other sites. The potential of nettle aphids Microlophium carnosum and their predator Anthocoris nemorum to disperse conidia of B. bassiana from soil to nettles and from sporulating cadavers in the nettle canopy was investigated in laboratory experiments. In petri dish assays, aphids showed potential to distribute B. bassiana from soil to nettle leaves. Predators dispersed inoculum from both soil and cadavers to nettle leaves in petri dishes. In microcosms, aphids did not disperse B. bassiana from the soil or from cadavers confined in the canopy, but A. nemorum were able to transfer inoculum from soil into the nettle canopy and to distribute conidia from cryptic cadavers. In some instances, infections were initiated in aphids and predators as a consequence of dispersal.     

Biological control of Strophosoma spp. (Coleoptera: Curculionidae) in greenery (Abies procera) plantations using Hyphomycetes

In Abies procera plantations Strophosoma melanogrammum and S. capitatum cause economic damage due to the adult stage feeding on the needles. No chemical treatments of these weevils are allowed in Denmark, so the potential of biological control was evaluated. We studied pathogenicity of thirteen isolates of entomopathogenic fungi and the field effect of soil application of Metarhizium anisopliae against Strophosoma spp. All of the tested isolates were capable of causing infections under laboratory conditions and average survival time at 20°C ranged between 13 and 23 days for S. melanogrammum and between 23 and 28 days for S. capitatum when dipped in a fungal suspension adjusted to 1×10⁷ conidia mL^-1. Under field conditions up to 90% of the living collected individuals died due to M. anisopliae infection in the treated plots, whereas less than 1% died of M. anisopliae infection in control plots. In accordance, the population of Strophosoma spp. was reduced in plots where the fungus was applied compared to control plots.     

Entomophthora ferdinandii (Zygomycetes: Entomophthorales) Causing Natural Infections of Musca domestica (Diptera: Muscidae) in Argentina

The identity and activity of an entomopathogenic fungus belonging to the Entomophthora muscae species complex and infecting Musca domestica in poultry houses from La Plata, Argentina, is reported. Entomophthora caused natural infections between September 2001 and September 2003. Primary conidia of this fungus were on average 29.5 ± 1.2 × 23.4 ± 2.4 μm and contained, on average, 10.5 ± 0.1 nuclei (range: 7–15) with an average diameter of 4.8 ± 0.1 μm. This fungus is identified as E. ferdinandii Keller (this specific epithet includes a nomenclaturally required spelling correction); this is a first record of E. ferdinandii in South America and of any member of the E. muscae species complex from flies in Argentina.     

Regulation of squalene epoxidase activity and comparison of catalytic properties of rat liver and Chinese hamster ovary cell-derived enzymes

Squalene epoxidase activity has been studied in cell-free preparations of Chinese hamster ovary (CHO) cells and rat liver. In contrast to rat liver microsomal squalene epoxidase, the enzyme of CHO cells is only slightly activated by the autologous cytosolic fraction, whereas phosphatidylglycerol or rat liver cytosolic preparations are potent stimulators of this enzyme. Triton X-100, a known stimulator of the hepatic squalene epoxidase, has no activating effect on the enzyme of CHO cells. The squalene epoxidase activity of both rat liver and CHO cells varies significantly according to the lipid content of the growth medium or diet. The changes in enzyme activity are shown to be entirely due to altered microsomal enzyme per se and not to changes in the activating properties of the soluble fraction. These results further support the proposed regulatory role of squalene epoxidase in cholesterogenesis.