Entomopathogenic fungus Beauveria bassiana-based bioinsecticide suppresses severity of powdery mildews of vegetables by inducing the plant defense responses.

The entomopathogenic fungus Beauveria bassiana is used commercially as a microbial insecticides against a wide range of agricultural insect pests. Some strains of B. bassiana protect the plants from pathogens, but the underlying mechanisms are largely unknown. Here, we found that prophylactic sprays of commercial bioinsecticide Botanigard on cucumber, tomato, and strawberry plants suppressed the severity of economically damaging powdery mildews. On leaf surfaces, hyphal elongation and spore germination of cucumber powdery mildew, Podosphaera xanthii, were inhibited, but B. bassiana strain GHA, the active ingredient isolated from Botanigard, only inhibited hyphal elongation but had no effect on spore germination of P. xanthii. In addition, strain GHA suppressed powdery mildew symptoms locally, not systemically. Treatment with Botanigard and strain GHA induced a hypersensitive response (HR)-like cell death in epidermal cells of the cucumber leaves in a concentration-dependent manner and inhibited penetration by P. xanthii. Transcriptome analysis and mass spectrometry revealed that GHA induced expression of salicylic acid (SA)-related genes, and treatment with Botanigard and GHA increased the SA level in the cucumber leaves. In NahG-transgenic tomato plants, which do not accumulate SA, the biocontrol effect of tomato powdery mildew by GHA was significantly reduced. These results suggested that B. bassiana GHA induces SA accumulation, leading to the induction of HR-like cell death against powdery mildew and subsequent suppression of fungal penetration. Thus, Botanigard has the potential to control both insect pests and plant diseases.

Entomopathogenic fungus Akanthomyces muscarius (Hypocreales: Cordycipitaceae) strain IMI 268317 colonises on tomato leaf surface through conidial adhesion and general and microcycle conidiation.

The entomopathogenic fungus Akanthomyces muscarius strain IMI 268317, previously known as Lecanicilliummuscariumand Verticillium lecanii, is currently used as a microbial insecticide to protect tomatoes from serious leaf-inhabiting pests in greenhouses. However, its persistence on tomato leaves has been unidentified. Understanding the events and processes of phyllosphere colonisation by this strain should help in developing its practical applications. This study assessed the epiphytic abilities of this strain on tomato leaves in humid conditions, simulating closed greenhouse environments. Conidia applied on tomato leaflets strongly adhered 12 h after inoculation. The mucilage-like materials were found around the germinated conidia after 3 days after inoculation (dpi), which possibly strengthened the adhesion. A total of 15% of conidia germinated at 3 dpi, of which 2% formed typical conidium or an enlarged structure on germ-tube tips. Many conidia were produced on phialide tips that branched from elongated hyphae at 7 dpi; however, invasion into leaf tissue was not observed. On the leaflets, inoculated conidia suspensions of 1 × 105 and 1 × 106 conidia/mL, colony forming units increased 52.6 and 8.8 folds from 0 to 14 dpi, respectively. These results suggested that A. muscarius strain IMI 268317 has high epiphytic abilities on tomato leaflets in a humid condition.

Biocontrol Activity of Nonpathogenic Strains of Fusarium oxysporum: Colonization on the Root Surface to Overcome Nutritional Competition.

Fusarium oxysporum is a soil-borne fungal pathogen that causes vascular wilts in a wide variety of crops. Certain nonpathogenic strains of F. oxysporum are known to protect crops against F. oxysporum pathogens. We assessed the biocontrol activities of nonpathogenic mutants of F. oxysporum ff. spp. melonis and lycopersici generated by disruption of the FOW2 gene, which encodes a Zn(II)2Cys6-type transcriptional regulator essential for their pathogenicity. Pre-inoculation of melon or tomato roots with strain ΔFOW2 conidia markedly reduced disease incidence caused by the parental wild-type strain in a concentration-dependent manner of conidial suspensions of ΔFOW2 strains. The biocontrol effect caused by the ΔFOW2 pre-inoculation lasted for at least 7 days. Pre-inoculation of melon roots with the wild-type or ΔFOW2 strain of F. oxysporum f. sp. lycopersici and nonpathogenic F. oxysporum strain also led to biocontrol activity against F. oxysporum f. sp. melonis, indicating that the biocontrol activity of ΔFOW2 strains is due to its nonpathogenic nature, not to the FOW2 disfunction. Conidial germination and hyphal elongation of only the wild-type strain were inhibited on melon root surface pre-inoculated with conidia of strains nonpathogenic to melon plants. Expression of defense-related genes was not significantly induced in roots and aboveground parts of melon seedlings preinoculated with ΔFOW2 conidia. Carbon source competition assay showed that nonpathogenic strains competed with the wild-type strain for a carbon source in soil. Strain ΔFOW2 also competed with the oomycete pathogen Pythium aphanidermatum for carbon source and protected melon plants from P. aphanidermatum. Our results suggest that the biocontrol activity of the nonpathogenic F. oxysporum strains used in this study mainly depends on their extensive colonization of the root surface and outcompeting pathogens for nutrients.

Identification and characterization of three microsporidian genera concurrently infecting a silkworm, Bombyx mori, in Brazil.

Microsporidia are important entomopathogens known for infecting insects such as the silkworm (Bombyx mori) thus impairing global silk production. This study aimed to identify and characterize the microsporidia isolated from a diseased larva of silkworm, collected from a sericulture farm in southern Brazil. Identification was performed by phylogenetic analysis of the nucleotide sequences of the SSU rRNA genes. Characterization was performed by analyzing spore sizes, tissue tropism, internal and external symptoms, and pathogenicity against B. mori. Microsporidia belonging to three different genera were identified, namely, Endoreticulatus, Nosema and Tubulinosema. After inoculation of the mixed spores of the microsporidian isolates into B. mori larvae, a high prevalence of Tubulinosema spp. was observed. This isolate showed high prevalence on the silk glands and a late mortality, initially of around 10% until the 20th day post-inoculation but reaching 91.5% upon pupation. Therefore, we demonstrated that Tubulinosema spp. causes chronic infection with slow pathogenicity. We identified for the first time three different microsporidians concurrently infecting B. mori in Brazil. Tubulinosema is of particular interest because of its potential threat to silk production; it affects the formation of silk glands in B. mori while not presenting distinguishable external symptoms or causing the immediate death of these insects. Further studies focusing on this species, mainly regarding its life cycle within the host and the sublethal effects of surviving individuals, demonstrate the importance of describing it as a new species and improving the characterization of the disease in order to prevent its spread.

Epiphytic and endophytic colonisation of tomato plants by the entomopathogenic fungus Beauveria bassiana strain GHA.

Beauveria bassiana, known for its entomopathogenic characteristics, is the most widely used biocontrol agent against many insect pests and may also be active against soil-borne pathogens. It inhabits the surfaces or inner tissues of various plant species without causing any visible signs or symptoms. Here we show that B. bassiana strain GHA, the active ingredient of a commercial microbial insecticide, colonises tomato plants. GHA grew on intact leaf surfaces of tomato in high humidity, but never entered stomata. Viable hyphae and conidia were detected, and the population on inoculated leaves significantly increased until 14 days after inoculation. On tomato leaves, GHA conidiated normally via conidiophores and phialides, and also via microcycle conidiation (conidiophores and phialides form directly from germ tubes and produce conidia). Hyphae were also detected inside the rachis, even more frequently after plant surfaces were scarified. These results suggested that B. bassiana strain GHA can grow epiphytically and endophytically on tomato plants.

Isolation of Metarhizium spp. from rhizosphere soils of wild plants reflects fungal diversity in soil but not plant specificity.

Understanding the abundance and diversity of fungal entomopathogens associated with plant species is critical for improving their field efficacy as microbial insecticides. Metarhizium is a cosmopolitan entomopathogenic fungus, with some species in this genus showing rhizosphere competencies. This study sought to determine the abundance and diversity of Metarhizium spp. in rhizosphere soils of wild plants in a field in Japan. Metarhizium spp. were detected in 76.2% of 151 rhizosphere soil samples collected from 41 plant species using a plating method. The mean density of Metarhizium spp. in rhizosphere soils was 1.2 × 104 colony forming units/g soil [base 10 logarithm of the mean = 4.06 (S.D. = 0.88)]. There was no significant difference in the densities and detection rates between Asteraceae and Poaceae as well as among two aster and one grass species. The fungal isolates were identified as five species, of which M. pingshaense was the most frequently detected and abundant species. No significant specific associations were recognised between the isolated Metarhizium spp. and the examined aster and grass species. Our findings demonstrated the high occurrence and abundance of M. pingshaense in rhizosphere soils of wild plants at the sampling site irrespective of host plant taxa.

Species associations and distributions of soil entomopathogenic fungi Metarhizium spp. in Japan.

Metarhizium Sorokin (Hypocreales: Clavicipitaceae) is a genus of facultative parasites of insects found in soils from various environments and is used for pest management. Understanding the habitat selection of Metarhizium spp. is critical to improve the efficacy and persistence of these fungi as microbial insecticides. This study sought to determine the habitat preferences of Metarhizium spp. in Japan. We identified 302 isolates of Metarhizium spp. as eight species by a combination of PCR-RFLP and phylogenetic analysis of DNA sequences. M. pingshaense was the predominant species in Japan and was most frequently isolated from both forest and agricultural environments. On the other hand, M. brunneum and M. pemphigi were comparatively restricted to forest environments. A similar species association was detected in a small area that was intensively investigated, where 7 species including 14 genotypes were isolated from soil. The results of this study have revealed different habitat preferences among Metarhizium spp. in Japan.

Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system.

Mutation and polymorphism detection by nucleases has become a more important tool in clinical and biological researches. There are several kinds of single-stranded nucleases for detecting mismatched DNAs. One of them, CEL II, was isolated from Apium graveolens and cleaves DNA with high specificity at sites of mismatch. High-throughput mutation scanning requires large quantity of CEL II endonuclease. Here, we demonstrate high-level expression of CEL II using silkworm-baculovirus system. The recombinant CEL II secreted in silkworm hemolymph was glycosylated and susceptible to N-glycosidase F. Additionally, larger metal ions such as Ca(2+) and Sr(2+) were able to replace Mg(2+) and enhanced mismatch cleavage activity of CEL II. These results indicate that the silkworm-baculovirus platform is a good alternative system to obtain the functional CEL II.