Comparative RNAseq Analysis of the Insect-Pathogenic Fungus <i>Metarhizium anisopliae</i> 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

Abiotic and Biotic Factors Affecting Resting Spore Formation in the Mite Pathogen Neozygites floridana

Neozygites floridana is an obligate mite pathogenic fungus in the Entomophthoromycota. It has been suggested that resting spores of this fungus are produced as a strategy to survive adverse conditions. In the present study, possible mechanisms involved in the regulation of resting spore formation were investigated in the hosts Tetranychus urticae and Tetranychus evansi. Abiotic and biotic factors mimicking conditions that we, based on earlier field studies, thought might induce resting spores in temperate and tropical regions were tested with isolates from Norway and Brazil. A total of 42 combinations of conditions were tested, but only one induced the formation of a high number of resting spores in only one isolate. The Brazilian isolate ESALQ1420 produced a large number of resting spores (51.5%) in T. urticae at a temperature of 11 ∘ C, photoperiod of 10L:14D, and light intensity of 42-46 ( mol m −2 s −1 ) on nonsenescent plants (nondiapausing females). Resting spores of the Brazilian N. floridana isolate ESALQ1421 were found at very low levels (up to 1.0%). Small percentages of T. urticae with resting spores (0-5.0%) were found for the Norwegian isolate NCRI271/04 under the conditions tested. The percentages of resting spores found for the Norwegian isolate in our laboratory studies are similar to the prevalence reported in earlier field studies

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Neozygites sp., é considerado como um dos mais importantes inimigos naturais do ácaro verde da mandioca, Mononychellus tanajoa (Bondar), no Nordeste do Brasil. A viabilização do uso deste patógeno em campo requer o conhecimento da sua distribuição geográfica, de métodos de produção"in vivo"e"in vitro"e o entendimento das relações patógeno-hospedeiro, abordados neste estudo. Neozygites sp. foi constatado nos estados de Alagoas, Bahia, Ceará, Paraíba, Pernambuco, Piauí e Sergipe. Ácaros infectados pelo fungo foram observados em 31 ,5% dos campos avaliados, predominantemente nas coletas realizadas durante os períodos úmidos, embora também tenham sido constatados ocasionalmente durante épocas secas. Estabeleceu-se um método para produção de Neozygites sp. utilizando-se como unidades de multiplicação discos de folha de mandioca onde foi realizado o contágio entre ácaros mumificados pelo patógeno e ácaros sadios. Crescimento de protoplastos de Neozygites sp., foi obtido em Meio de Grace + 0,35g/I de NaHCO3 e no Meio 199 (com sais de Hank) + 26,68g/I de sacarose + 2,2g/l de NaHCO3 , quando foram enriquecidos com soro fetal bovino, às proporções de 5 ou 8% do volume total. A ocorrência do patógeno foi estudada no período de março a outubro de 1 994 em 9 campos de mandioca em Piritiba - Bahia para o conhecimento de detalhes da epizootia. O período entre o início do surgimento de ácaros infectados em um dos campos, até à constatação de ácaros infectados em todos os campos durante a epizootia foi de 23 dias. A determinação da existência de uma variabilidade espaço-temporal na ocorrência inicial de Neozygites sp., em campos de mandioca, indica a conveniência da transferência do patógeno de um campo a outro para antecipar o início de epizootias em campos diferentes. Reduções nas populações de M. tanajoa estiveram correlacionadas com incrementos nos níveis de infecção por Neozygites sp., durante os meses de junho e julho. As epizootias foram caracterizadas por um curto espaço de tempo entre o surgimento do patógeno e a rápida redução nas altas densidades populacionais de M. tanajoa. Na fase pós-epizoótíca a densidade de M. tanajoa permaneceu por algum tempo em níveis muito reduzidos, diminuindo consequentemente a incidência do patógeno em campo. O surgimento de novos casos da doença foi proporcionalmente maior nas plantas com maior densidade populacional de M. tanajoa dentro de um mesmo campo. Ácaros infectados foram observados em maior proporção nas folhas apicais. Durante os meses de junho e julho ocorreu substancial formação de esporos de resistência. É possível que esta fase do ciclo biológico seja a principal forma em que o patógeno transponha os períodos de seca no Nordeste do Brasilnot availabl

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Neozygites sp., é considerado como um dos mais importantes inimigos naturais do ácaro verde da mandioca, Mononychellus tanajoa (Bondar), no Nordeste do Brasil. A viabilização do uso deste patógeno em campo requer o conhecimento da sua distribuição geográfica, de métodos de produção"in vivo"e"in vitro"e o entendimento das relações patógeno-hospedeiro, abordados neste estudo. Neozygites sp. foi constatado nos estados de Alagoas, Bahia, Ceará, Paraíba, Pernambuco, Piauí e Sergipe. Ácaros infectados pelo fungo foram observados em 31 ,5% dos campos avaliados, predominantemente nas coletas realizadas durante os períodos úmidos, embora também tenham sido constatados ocasionalmente durante épocas secas. Estabeleceu-se um método para produção de Neozygites sp. utilizando-se como unidades de multiplicação discos de folha de mandioca onde foi realizado o contágio entre ácaros mumificados pelo patógeno e ácaros sadios. Crescimento de protoplastos de Neozygites sp., foi obtido em Meio de Grace + 0,35g/I de NaHCO3 e no Meio 199 (com sais de Hank) + 26,68g/I de sacarose + 2,2g/l de NaHCO3 , quando foram enriquecidos com soro fetal bovino, às proporções de 5 ou 8% do volume total. A ocorrência do patógeno foi estudada no período de março a outubro de 1 994 em 9 campos de mandioca em Piritiba - Bahia para o conhecimento de detalhes da epizootia. O período entre o início do surgimento de ácaros infectados em um dos campos, até à constatação de ácaros infectados em todos os campos durante a epizootia foi de 23 dias. A determinação da existência de uma variabilidade espaço-temporal na ocorrência inicial de Neozygites sp., em campos de mandioca, indica a conveniência da transferência do patógeno de um campo a outro para antecipar o início de epizootias em campos diferentes. Reduções nas populações de M. tanajoa estiveram correlacionadas com incrementos nos níveis de infecção por Neozygites sp., durante os meses de junho e julho. As epizootias foram caracterizadas por um curto espaço de tempo entre o surgimento do patógeno e a rápida redução nas altas densidades populacionais de M. tanajoa. Na fase pós-epizoótíca a densidade de M. tanajoa permaneceu por algum tempo em níveis muito reduzidos, diminuindo consequentemente a incidência do patógeno em campo. O surgimento de novos casos da doença foi proporcionalmente maior nas plantas com maior densidade populacional de M. tanajoa dentro de um mesmo campo. Ácaros infectados foram observados em maior proporção nas folhas apicais. Durante os meses de junho e julho ocorreu substancial formação de esporos de resistência. É possível que esta fase do ciclo biológico seja a principal forma em que o patógeno transponha os períodos de seca no Nordeste do Brasilnot availabl

Colletotrichum nymphaeae var. entomophilum var. nov. a natural enemy of the citrus scale insect, Praelongorthezia praelonga (Hemiptera: Ortheziidae)

The citrus scale insect Praelongorthezia praelonga (Douglas), a major pest of citrus and other economically important crops, has only two commonly documented natural enemies: an entomopathogenic strain of the fungus Colletotrichum nymphaeae (Pass.) Aa and several parasitoids. The entomopathogenic strain of C. nymphaeae, formerly recognized under the synonym C. gloeosporioides f. sp. ortheziidae, is under development for commercial application as a biological control agent in citrus in Brazil-the top exporter of citrus globally. The synonomy of C. gloeosporioides f. sp. ortheziidae with C. nymphaeae remains based on limited DNA sequence data and without morphological study. To qualify for future approval as a biological control agent by federal agencies in Brazil and the European Union, the circumscription of a microorganism must be explicit and without ambiguities. Herein, through morphological study and phylogenetic analysis of five DNA regions we clarify the circumscription and affinity of entomopathogenic C. nymphaeae and describe it as a new variety

Cereal straw mulching in strawberry—A facilitator of plant visits by edaphic predatory mites at night?

In Norway, strawberry producers use cereal straw mulching to prevent berries from contacting the soil and to control weeds. We hypothesized that organic matter such as straw mulch also favors the maintenance of predatory mites which visit strawberry plants at nighttime. We compared mite diversity in cereal straw exposed for different periods in strawberry fields and evaluated their possible migration to plants in two experiments with potted plants in 2019. An ‘Early season’ experiment compared no mulching (T1), oat straw mulch exposed in field since 2018 (T2), or 2017 (T3), while a ‘Mid-season’ experiment compared no mulching (T1), barley straw mulch from 2018 (T2), or a mix from 2017 and 2018 (T3). To provide edaphic predatory mites with a potential source of food, all plants were infested with two-spotted spider mite (Tetranychus urticae Koch). Results suggested that straw mulch facilitates the prevalence of predatory mites in strawberry fields. Most predatory mite visits were at night, confirming our initial hypothesis. Predominant nocturnal mites on leaves belonged to Melicharidae (Proctolaelaps sp.) (‘Early season’, T2), Blattisociidae (Lasioseius sp.) (‘Early and Mid-season’, T3) and Phytoseiidae (‘Mid-season’, T2). Parasitus consanguineus Oudemans & Voigts was the predominant species (‘Early season’, T3) at the base of plants. Anystidae were diurnal visitors only (‘Mid-season’, T2). Future studies should evaluate the predation potential of Proctolaelaps sp. and Lasioseius sp. on two-spotted spider mite and other strawberry pests.publishedVersio

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.publishedVersio

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.acceptedVersio