Genomic signatures and insights into host niche adaptation of the entomopathogenic fungus Metarhizium humberi.

The genus Metarhizium is composed of species used in biological control programs of agricultural pests worldwide. This genus includes common fungal pathogen of many insects and mites and endophytes that can increase plant growth. Metarhizium humberi was recently described as a new species. This species is highly virulent against some insect pests and promotes growth in sugarcane, strawberry, and soybean crops. In this study, we sequenced the genome of M. humberi, isolate ESALQ1638, and performed a functional analysis to determine its genomic signatures and highlight the genes and biological processes associated with its lifestyle. The genome annotation predicted 10633 genes in M. humberi, of which 92.0% are assigned putative functions, and ∼17% of the genome was annotated as repetitive sequences. We found that 18.5% of the M. humberi genome is similar to experimentally validated proteins associated with pathogen-host interaction. Compared to the genomes of eight Metarhizium species, the M. humberi ESALQ1638 genome revealed some unique traits that stood out, e.g., more genes functionally annotated as polyketide synthases (PKSs), overrepresended GO-terms associated to transport of ions, organic and amino acid, a higher percentage of repetitive elements, and higher levels of RIP-induced point mutations. The M. humberi genome will serve as a resource for promoting studies on genome structure and evolution that can contribute to research on biological control and plant biostimulation. Thus, the genomic data supported the broad host range of this species within the generalist PARB clade and suggested that M. humberi ESALQ1638 might be particularly good at producing secondary metabolites and might be more efficient in transporting amino acids and organic compounds.

Comparative RNAseq Analysis of the Insect-Pathogenic Fungus Metarhizium anisopliae Reveals Specific Transcriptome Signatures of Filamentous and Yeast-Like Development.

The fungus Metarhizium anisopliae is a facultative insect pathogen used as biological control agent of several agricultural pests worldwide. It is a dimorphic fungus that is able to display two growth morphologies, a filamentous phase with formation of hyphae and a yeast-like phase with formation of single-celled blastospores. Blastospores play an important role for M. anisopliae pathogenicity during disease development. They are formed solely in the hemolymph of infected insects as a fungal strategy to quickly multiply and colonize the insect's body. Here, we use comparative genome-wide transcriptome analyses to determine changes in gene expression between the filamentous and blastospore growth phases in vitro to characterize physiological changes and metabolic signatures associated with M. anisopliae dimorphism. Our results show a clear molecular distinction between the blastospore and mycelial phases. In total 6.4% (n = 696) out of 10,981 predicted genes in M. anisopliae were differentially expressed between the two phases with a fold-change > 4. The main physiological processes associated with up-regulated gene content in the single-celled yeast-like blastospores during liquid fermentation were oxidative stress, amino acid metabolism (catabolism and anabolism), respiration processes, transmembrane transport and production of secondary metabolites. In contrast, the up-regulated gene content in hyphae were associated with increased growth, metabolism and cell wall re-organization, which underlines the specific functions and altered growth morphology of M. anisopliae blastospores and hyphae, respectively. Our study revealed significant transcriptomic differences between the metabolism of blastospores and hyphae. These findings illustrate important aspects of fungal morphogenesis in M. anisopliae and highlight the main metabolic activities of each propagule under in vitro growth conditions.

Mulching with coffee husk and pulp in strawberry affects edaphic predatory mite and spider mite densities.

Mulching of soil beds of strawberry fields is usually done with polyethylene film in southern Minas Gerais state, Brazil. This material is relatively expensive and difficult to discard after use. In some countries, mulching is done with the use of organic material that could have an advantage over the use of plastic for its easier degradation after use, and for favoring edaphic beneficial organisms. Predatory mites (especially Gamasina, Mesostigmata) may be abundant in the soil and could conceivably move to the soil surface and onto the short-growing strawberry plants at night, helping in the control or pest arthropods. The two-spotted spider mite, Tetranychus urticae Koch, is considered an important strawberry pest in that region, where the fungus Neozygites floridana (Weiser and Muma) has been found to infect it. Different mulching types could affect the incidence of this pathogen. Dehydrated coffee husk and pulp (DCHP) is a byproduct readily available in southern Minas Gerais, where could be used as organic mulching in strawberry beds. The temporary contact of that material with the soil of a patch of natural vegetation could facilitate its colonization by edaphic predatory mites helpful in the control of strawberry pests. The objective of this work was to study the effect of mulching type on the population dynamics of the two-spotted spider mite, associate mites and N. floridana, in a greenhouse and in the field. The use of DCHP increased the number of edaphic Gamasina on strawberry plants-Proctolaelaps pygmaeus (Müller) (Melicharidae) and Blattisocius dentriticus (Berlese) (Blattisociidae) were observed on strawberry leaflets, mainly in nocturnal samplings, indicating their possible daily migration from soil to plants. Lower levels of two-spotted spider mite occurred on plants from pots or soil beds mulched with DCHP instead of polyethylene film, possibly because of the slightly higher levels of mites of the family Phytoseiidae and infection by N. floridana. Adding DCHP onto the floor of natural vegetation did not result in higher diversity or levels of gamasine mites on DCHP. Complementary studies should be conducted to find ways to increase diversity and density of those organisms in strawberry beds, in an attempt to improve biological control of strawberry pests. The decision to use DCHP for mulching should also take into account other factors such as strawberry yield, costs and efficiency of weed management, to be evaluated in subsequent studies.

Modified Adamek's medium renders high yields of Metarhizium robertsii blastospores that are desiccation tolerant and infective to cattle-tick larvae.

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.

Improved shelf life of dried Beauveria bassiana blastospores using convective drying and active packaging processes.

The yeast form (blastospore) of the dimorphic insect-pathogenic fungus Beauveria bassiana can be rapidly produced using liquid fermentation methods but is generally unable to survive rapid dehydration processes or storage under non-refrigerated conditions. In this study, we evaluated the influence of two convective drying methods, various modified atmosphere packaging systems, and storage temperatures on the desiccation tolerance, storage stability, and virulence of blastospores of B. bassiana ESALQ 1432. All blastospore formulations were dried to <5 % water content equivalent to aw < 0.3. The viability of B. bassiana blastospores after air drying and spray drying was greater than 80 %. Vacuum-packaged blastospores remained viable longer when stored at 4 °C compared with 28 °C with virtually no loss in viability over 9 months regardless the drying method. When both oxygen and moisture scavengers were added to sealed packages of dried blastospore formulations stored at 28 °C, viability was significantly prolonged for both air- and spray-dried blastospores. The addition of ascorbic acid during spray drying did not improve desiccation tolerance but enhanced cell stability (∼twofold higher half-life) when stored at 28 °C. After storage for 4 months at 28 °C, air-dried blastospores produced a lower LC80 and resulted in higher mortality to whitefly nymphs (Bemisia tabaci) when compared with spray-dried blastospores. These studies identified key storage conditions (low aw and oxygen availability) that improved blastospore storage stability at 28 °C and will facilitate the commercial development of blastospores-based bioinsecticides.

Life history of the predatory mite Phytoseiulus fragariae on Tetranychus evansi and Tetranychus urticae (Acari: Phytoseiidae, Tetranychidae) at five temperatures.

Tetranychus evansi Baker and Pritchard and Tetranychus urticae Koch (Acari: Tetranychidae) are important pests of Solanaceae in many countries. Several studies have demonstrated that T. urticae is an acceptable prey to many predatory mites, although the suitability of this prey depends on the host plant. T. evansi, has been shown to be an unfavorable prey to most predatory mites that have been tested against it. The predator Phytoseiulus fragariae Denmark and Schicha (Acari: Phytoseiidae) has been found in association with the two species in Brazil. The objective of this work was to compare biological parameters of P. fragariae on T. evansi and on T. urticae as prey. The study was conducted under laboratory conditions at 10, 15, 20, 25 and 30 degrees C. At all temperatures, survivorship was lower on T. evansi than on T. urticae. No predator reached adulthood at 10 degrees C on the former species; even on the latter species, only about 36% of the predators reached adulthood at 10 degrees C. For both prey, in general, duration of each life stage was shorter, total fecundity was lower and intrinsic rate of population increase (r(m)) was higher with increasing temperatures. The slower rate of development of P. fragariae on T. evansi resulted in a slightly higher thermal requirement (103.9 degree-days) on that prey than on T. urticae (97.1 degree-days). The values of net reproduction rate (R (0)), intrinsic rate of increase (r(m)) and finite rate of increase (lambda) were significantly higher on T. urticae, indicating faster population increase of the predator on this prey species. The highest value of r(m) of the predator was 0.154 and 0.337 female per female per day on T. evansi and on T. urticae, respectively. The results suggested that P. fragariae cannot be considered a good predator of T. evansi.