Conidial Yield Research Articles

O-mannosyltransferase MaPmt2 contributes to stress tolerance, cell wall integrity and virulence in Metarhizium acridum

As a conserved post-translational modification, O-mannosyltransferase families play important roles in many cellular processes. Three subfamilies (MaPmt1, MaPmt2 and MaPmt4) are grouped in Metarhizium acridum according to sequence homology. The functions of MaPmt1 and MaPmt4 have been characterized in M. acridum previously. In this study, the functions of another member belonging to the Pmt2 subfamily, MaPmt2, were identified through RNAi strategy. The three MaPmt2 knockdown mutants showed dramatically decreased expression of MaPmt2. Phenotypic analyses showed that the mutants exhibited decreased tolerances to wet-heat, UV-B irradiation and cell wall perturbing chemicals. Further studies revealed that the mutants presented thinner cell walls observed by transmission electron microscope combined with changed cell wall components. Besides, knockdown of MaPmt2 decelerated conidial germination and decreased conidial yield. Compared with the wild-type strain, the MaPmt2 knockdown mutants caused impaired virulence only by topical inoculation. Results illustrated that the decreased virulence by inoculation could result from the delayed conidial germination on locust wings, reduced appressorium formation, as well as reduced turgor pressure in MaPmt2 knockdown mutants.

BbWor1, a Regulator of Morphological Transition, Is Involved in Conidium-Hypha Switching, Blastospore Propagation, and Virulence in Beauveria bassiana.

ABSTRACTMorphological transition is an important adaptive mechanism in the host invasion process. Wor1 is a conserved fungal regulatory protein that controls the phenotypic switching and pathogenicity of Candida albicans. By modulating growth conditions, we simulated three models of Beauveria bassiana morphological transitions, including CTH (conidia to hyphae), HTC (hyphae to conidia), and BTB (blastospore to blastospore). Disruption of BbWor1 (an ortholog of Wor1) resulted in a distinct reduction in the time required for conidial germination (CTH), a significant increase in hyphal growth, and a decrease in the yield of conidia (HTC), indicating that BbWor1 positively controls conidium production and negatively regulates hyphal growth in conidium-hypha switching. Moreover, ΔBbWor1 prominently decreased blastospore yield, shortened the G0/G1 phase, and prolonged the G2/M phase under the BTB model. Importantly, BbWor1 contributed to conidium-hypha switching and blastospore propagation via different genetic pathways, and yeast one-hybrid testing demonstrated the necessity of BbWor1 to control the transcription of an allergen-like protein gene (BBA_02580) and a conidial wall protein gene (BBA_09998). Moreover, the dramatically weakened virulence of ΔBbWor1 was examined by immersion and injection methods. Our findings indicate that BbWor1 is a vital participant in morphological transition and pathogenicity in entomopathogenic fungi.IMPORTANCE As a well-known entomopathogenic fungus, Beauveria bassiana has a complex life cycle and involves transformations among single-cell conidia, blastospores, and filamentous hyphae. This study provides new insight into the regulation of the fungal cell morphological transitions by simulating three models. Our research identified BbWor1 as a core transcription factor of morphological differentiation that positively regulates the production of conidia and blastospores but negatively regulates hyphal growth. More importantly, BbWor1 affects fungal pathogenicity and the global transcription profiles within three models of growth stage transformation. The present study lays a foundation for the exploration of the transition mechanism of entomopathogenic fungi and provides material for the morphological study of fungi.

Effect of Osmotic Stress on the Growth, Development and Pathogenicity of Setosphaeria turcica.

Osmotic stress is a severe condition frequently encountered by microorganisms; however, there is limited knowledge on the influence of hyperosmotic stress on the growth, development and pathogenicity of phytopathogenic fungi. Here, three osmotic conditions (0.4 M NaCl, 0.4 M KCl, and 0.6 M sorbitol supplemented in potato dextrose agar medium) were used to identify the effect of osmotic stress on the growth, development and pathogenicity of Setosphaeria turcica which is a plant pathogenic fungus and causes northern corn leaf blight disease in maize, sorghum, and related grasses. In osmotic stress, the growth rate of mycelium was decreased, and the number of vesicular structures and flocculent secretion outside the hypha cell wall were significantly increased. The qRT-PCR results showed that the osmotic stress quickly activated the HOG-MAPK pathway, up-regulated the expression of the downstream genes, and these genes were most highly expressed within 30 min of exposure to osmotic stress. Furthermore, the germination rate and the yield of conidia were significantly higher under osmotic stress than in the control. A pathogenicity analysis confirmed that pathogenicity of the conidia which were cultured under osmotic stress was significantly enhanced. By analyzing the knock-out mutants of an osmotic stress responsed gene StFPS1, an aquaglyceroporin downstream of the HOG-MAPK pathway, we found that StFPS1 was involved in the formation of appressorium and penetration peg, which affected the penetration ability of S. turcica. In summary, our work explained the correlation between osmotic stress and growth, development, and pathogenicity in S. turcica.

MaAreB, a GATA Transcription Factor, Is Involved in Nitrogen Source Utilization, Stress Tolerances and Virulence in Metarhizium acridum.

The nitrogen catabolite repression (NCR) pathway is involved in nitrogen utilization, in which the global GATA transcription factor AreA plays an indispensable role and has been reported in many fungi. However, relatively few studies are focused on AreB, another GATA transcription factor in the NCR pathway and the functions of AreB are largely unknown in entomopathogenic fungi. Here, we characterized MaAreB in the model entomopathogenic fungus Metarhizium acridum. Sequence arrangement found that MaAreB had a conserved GATA zinc finger DNA binding domain and a leucine zipper domain. Disruption of MaAreB affected the nitrogen utilization and led to decelerated conidial germination and hyphal growth, decreased conidial yield, and lower tolerances to UV-B irradiation and heat-shock. Furthermore, the MaAreB mutant (ΔMaAreB) exhibited increased sensitivity to CFW (Calcofluor white), decreased cell wall contents (chitin and β-1,3-glucan) and reduced expression levels of some genes related to cell wall integrity, indicating that disruption of MaAreB affected the cell wall integrity. Bioassays showed that the virulence of the ΔMaAreB strain was decreased in topical inoculation but not in intra-hemocoel injection. Consistently, deletion of MaAreB severely impaired the appressorium formation and reduced the turgor pressure of appressorium. These results revealed that MaAreB regulated fungal nitrogen utilization, cell wall integrity and biological control potential, which would contribute to the functional characterization of AreB homologous proteins in other insect fungal pathogens, and even filamentous fungi.

Characterization of a new strain of Metarhizium novozealandicum with potential to be developed as a biopesticide

ABSTRACT The fungal species Metarhizium novozealandicum, that occurs only in New Zealand and Australia has been poorly studied. In this work, a new strain of M. novozealandicum isolated from a larva of Wiseana sp. is described based on morphology, genomic multilocus (ITS, EF-1α and β-tubulin) phylogeny, growth in different culture media and insecticidal activity. The isolate AgR-F177 was clustered in the same clade with M. novozealandicum. AgR-F177 colonies developed faster on Sabouraud Dextrose Agar (SDA) than on Potato Dextrose Agar (PDA) when incubated at 25°C, with no growth observed at 30°C on either media. Conidia yield on an oat-based medium in semisolid fermentation was 7.41 x 108conidia/g of substrate and a higher yield of 1.68 x 109conidia/g of substrate was obtained using solid fermentation on cooked rice. AgR-F177 formed microsclerotia (MS) in liquid fermentation after 7 days reaching the maximum yield of 3.3 × 103 MS/mL after 10 days. AgR-F177 caused mortality in Wiseana copularis, Costelytra giveni and Plutella xylostella larvae with efficacies up to 100%, 69.2%, and 45.7%, respectively. The ease of production of AgR-F177 with different fermentation systems and its pathogenicity against different insect pests reveal its potential as a new biopesticide.

The N-mannosyltransferase gene BbAlg9 contributes to cell wall integrity, fungal development and the pathogenicity of Beauveria bassiana

The mannosyltransferase Alg9 plays a vital role in N-linked protein glycosylation in Saccharomyces cerevisiae, but its function in most filamentous fungi is not clear. The present study characterized BbAlg9 (an ortholog of S. cerevisiae Alg9) in Beauveria bassiana to determine the roles of N-mannosyltransferase in biological control potential of the filamentous entomopathogenic fungus. The disruption of BbAlg9 led to slower fungal growth in media with various nutrition compositions. The conidiation of ΔBbAlg9 was less than that of the wild type from the third to the fifth day but showed no significant difference on the sixth day, suggesting that BbAlg9 affects the development of conidia rather than conidial yield of late stage. ΔBbAlg9 showed defects in conidial germination, multiple stress tolerances and the yield of blastospores, with altered size and density, and virulence in hosts infected via the immersion and injection methods. The deletion of BbAlg9 resulted in defects in cell wall integrity, including increased mannoprotein and glucan content and decreased chitin content, which were accompanied by transcriptional activation or suppression of genes related to cell wall component biosynthesis. Notably, deletion of the N-mannosyltransferase BbAlg9 altered the transcription levels of O-mannosyltransferase genes (Pmt and Ktr family). These data show that BbAlg9 is involved in the fungal development, conidial stress tolerance, cell wall integrity and virulence of B. bassiana.

Vital roles of Pks11, a highly reducing polyketide synthase, in fungal conidiation, antioxidant activity, conidial cell wall integrity, and UV tolerance of Beauveria bassiana

Fungal polyketide synthases play important and differential roles in synthesizing secondary metabolites and regulating several cell events, including asexual development, environmental adaptation, and pathogenicity. This study shows the important functions of a highly reducing polyketide synthase, Pks11, in Beauveria bassiana, a filamentous fungal insect pathogen used worldwide for pest biocontrol. The deletion of pks11 led to severe defects in conidial yields on different media and a decrease of 36.27% in the mean thickness of conidial cell wall under normal conditions. Compared with the wild-type, Δpks11 showed higher tolerance to oxidation and increased sensitivity to high temperature during colony growth. Moreover, the lack of pks11 caused a decrease in conidial germination after exposure to UV radiation but did not affect the virulence of B. bassiana against Galleria mellonella larvae via typical cuticle infection. These findings concurred with the alteration in the transcript levels of some phenotype-related genes. These data suggested that pks11 played vital roles in the asexual development, cell wall integrity, and fungal responses to oxidation, high temperature, and UV irradiation of B. bassiana.

Essential Roles of Two FRQ Proteins (Frq1 and Frq2) in Beauveria bassiana's Virulence, Infection Cycle, and Calcofluor-Specific Signaling.

Two FRQ proteins (Frq1 and Frq2) distinct in molecular mass and structure coexist in Beauveria bassiana, an asexual insect-pathogenic fungus. Frq1 and Frq2 have been proven to have opposite nuclear rhythms that can persistently activate developmental activator genes and hence orchestrate nonrhythmic conidiation in vitro under light or in darkness. Here, we report the essentiality of either FRQ, but Frq2 being more important than Frq1, for the fungal virulence and infection cycle. The fungal virulence was attenuated significantly more in the absence of frq2 than in the absence of frq1 through either normal cuticle infection or cuticle-bypassing infection by intrahemocoel injection, accompanied by differentially reduced secretion of Pr1 proteases required for the cuticle infection and delayed development of hyphal bodies in vivo, which usually propagate by yeast-like budding in the host hemocoel to accelerate insect death from mycosis. Despite insignificant changes in radial growth under normal, oxidative, and hyperosmotic culture conditions, conidial yields of the Δfrq1 and Δfrq2 mutants on insect cadavers were sharply reduced, and the reduction increased with shortening daylight length on day 9 or 12 after death, indicating that both Frq1 and Frq2 are required for the fungal infection cycle in host habitats. Intriguingly, the Δfrq1 and Δfrq2 mutants showed hypersensitivity and high resistance to cell wall-perturbing calcofluor white, coinciding respectively with the calcofluor-triggered cells' hypo- and hyperphosphorylated signals of Slt2, a mitogen-activated protein kinase (MAPK) required for mediation of cell wall integrity. This finding offers a novel insight into opposite roles of Frq1 and Frq2 in calcofluor-specific signal transduction via the fungal Slt2 cascade.IMPORTANCE Opposite nuclear rhythms of two distinct FRQ proteins (Frq1 and Frq2) coexisting in an asexual fungal insect pathogen have been shown to orchestrate the fungal nonrhythmic conidiation in vitro in a circadian day independent of photoperiod change. This paper reports essential roles of both Frq1 and Frq2, but a greater role for Frq2, in sustaining the fungal virulence and infection cycle since either frq1 or frq2 deletion led to marked delay of lethal action against a model insect and drastic reduction of conidial yield on insect cadavers. Moreover, the frq1 and frq2 mutants display hypersensitivity and high resistance to cell wall perturbation and have hypo- and hyperphosphorylated MAPK/Slt2 in calcofluor white-triggered cells, respectively. These findings uncover a requirement of Frq1 and Frq2 for the fungal infection cycle in host habitats and provide a novel insight into their opposite roles in calcofluor-specific signal transduction through the MAPK/Slt2 cascade.

Comparative study of fungal stability between Metarhizium strains after successive subculture

BackgroundMetarhizium species are considered one of the most outstanding powerful biological control agents that have been commercialized as biopesticides against various agricultural pests. Fungal stability with successive in vitro cultivation is a desirable trait for a large-scale production of fungal biopesticide.Main bodyThe new Egyptian strain Metarhizium anisopliae AUMC 3262 exhibited auspicious results when compared to Metarhizium brunneum ARSEF 4556 and M. brunneum V275 based on the variations of fungal characteristics, and essential quality control parameters (radial growth rate, conidial yield, viability, and virulence) after repeated in vitro subculturing. Changes in morphological characteristics were noted at both AUMC 3262 and ARSEF 4556. Following the 5th subculture, decreased conidial yield was noted, though radial growth remained stable, confirming that there is a non-positive correlation between conidial yield and radial growth rate for these species. In contrast, V275 showed a high morphological stability, conidial yield, and radial growth rate after repeated subculture. The three tested strains manifested high viability up to 100% and displayed the same pattern of Pr1 production. A slight variation was recorded in the median lethal time (LT50) values against the great wax moth, Galleria mellonella (L.), larvae between different subcultures of the tested Metarhizium strains.ConclusionThe new Egyptian strain AUMC 3262 showed a high stability with a slight difference in some parameters after the successive subculture compared to both ARSEF4556 and V275.

Production of conidia using different culture media modifies the virulence of the entomopathogenic fungus Metarhiziumys against Aedes aegypti larvae.

Entomopathogenic fungi are being investigated for the biological control of a range of mosquitoes. Metarhizium conidiospores (conidia) effectively kill Aedes aegypti larvae and could be deployed as an alternative to chemical insecticides. Conidial yield and virulence of fungi when cultured on three different types of solid media, was investigated. Three culture media were tested: a) Sabouraud dextrose agar (SDA); b) rice flour yeast agar (RYA) and c) rice grains. Conidia produced using these substrates were tested for virulence against Ae. aegypti larvae obtained from field collected eggs. Larvae (2nd - 3rd instar) were exposed to aqueous conidial suspensions and survival monitored over 7 days. Survival analysis was performed using Log-Rank and Kaplan Meier tests, while fungal growth and conidial yields were analyzed using a two-way ANOVA. There were only small differences between growth rates on RYA and SDA; however, ESALQ 818 showed the highest conidial yield on rice. Conidia produced on rice grains were more virulent, rapidly reducing survival rates of mosquito larvae. ESALQ 818 conidia produced on rice grains, RYA and SDA killed 100% of the larvae on the 2nd, 3rd and 4th day of exposure, respectively. IP 46 virulence of was consistently lower than ESALQ 818 for all the media tested. The choice of culture media can influence the virulence of fungal conidia to Ae. aegypti larvae, demonstrating the importance of not only selecting the most virulent isolate but also standardizing growth conditions when screening for virulence.

Expression of a phenoloxidase cascade inhibitor enhances the virulence of the fungus Beauveria bassiana against the insect Helicoverpa armigera

Entomopathogenic fungi have high potential for controlling insect pests, although the slow killing speed has blocked their widespread application. To increase the virulence of entomopathogenic fungi, genetic modification can be employed. Egf1.0 is an immunosuppressive protein encoded by polydnavirus, carried by parasitoid wasp Microplitis demolitor, which blocks the prophenoloxidase (PPO) activation response of host insects. In this study, we explored the feasibility of genetically modifying entomopathogenic fungi with increased virulence by expressing Egf1.0. In comparison with the wild-type parents, the median lethal concentration (LC50) of Beauveria bassiana expressing Egf1.0 against Helicoverpa armigera was reduced by 2.7-fold, and the median lethal time (LT50) was reduced by 22.8%. In vitro assay showed that recombinant Egf1.0 was able to inhibit the PPO activation response of H. armigera. In vivo assay revealed that the expression of Egf1.0 in B. bassiana caused a higher degree of suppression to PPO activation response of H. armigera. These assays suggested that the increased virulence of the transgenic fungi is due to the increased ability to suppress the host insect's immune response. Moreover, colony growth, conidia yield, and germination assays revealed that the expression of Egf1.0 in B. bassiana had no effect on its growth and development. In conclusion, the expression of Egf1.0 can significantly enhance the pathogenicity of B. bassiana against host insects.

G\u03b13 subunit Thga3 positively regulates conidiation, mycoparasitism, chitinase activity, and hydrophobicity of Trichoderma harzianum

Heterotrimeric G-proteins are key elements of signal transduction pathways, which participate in regulating multiple biological processes in fungi including growth, conidiation, antagonism, and mycoparasitism. Among G protein subunits, Gα3 showed diverse regulatory functions in different fungi. In this study, we cloned a Gα3 subunit coding gene thga3 from T. harzianum Th33 that can antagonize Rhizoctonia solani and some other plant pathogenic fungi. A thga3 deletion strain Δthga3 was generated using the double-crossover homologous recombination strategy, and Rthga3 was generated by transforming thga3-expressing vector into the protoplasts of Δthga3 by the PEG/CaCl2-mediated method. The biological characteristics of wild-type Th33, Δthga3 and Rthga3 were evaluated. Compared with wild-type Th33, Δthga3 showed 15%, 94%, and 23% decrease in hyphal growth, conidia yield, and chitinase activity, respectively, and Δthga3 showed lower antagonistic and mycoparasitism abilities, while there were no significant differences between wild-type Th33 and Rthga3. The hyphal surface hydrophobicity of Δthga3 significantly decreased compared with those of the wild-type Th33 and Rthga3. qRT-PCR analysis revealed that transcript abundance of the hydrophobin gene (tha_09745) of Δthga3 decreased by 80% compared with that of wild-type Th33 and Rthga3. The results showed that thga3 positively regulates the growth, conidiation, hydrophobicity, chitinase activities, and mycoparasitism of Th33 towards R. solani. We hence deduced that the expression level of Tha_09745 is correlated to the hyphal hydrophobicity of Th33 and therefore affects the other biological characteristics of Th33. The findings of this report provide a foundation for elucidating the G-protein signal regulatory mechanisms of fungi.

Long-lasting effects of red and blue light exposure on the growth of soil fungi

The experimental assessment of inter-species difference in long lasting effects produced in fungi by a brief exposure to the monochromatic light was performed. 24-h cultures grown from 1 mm mycelium fragments of Alternaria alternata, Aspergillus clavatus, Fusarium fujikuroi, Penicillium citrinum and Trichoderma viride were exposed for 30 min to blue light (BL, 450 nm) or red light (RL, 660 nm) and cultured for the next 10 days. Radial growth rate, conidial yield and germination, contents of proteins and phenolsand fungal antibacterial activity were estimated. BL- or RL-exposure did not essentially affect the final size of colonies of A. clavatus but delayed the growth of P. citrinum and stimulated it in A. alternata and F. fujikuroi; these changes were more profound after BL, than after RL. In T. viride the BL exposure led to a remarkable delay of growth, whereas the RL significantly increased the growth rate. Photo-induced changes in the conidial yield, conidial germination, contents of proteins and phenols also were dependent on the light wavelength and showed strong inter-species heterogeneity. Fungal antibacterial activity in exposed cultures was similar to the unexposed control. The observed effects are indicative targets for future research of possible molecular regulatory mechanisms underlying the photobiology in different fungal taxons.

MaPmt1, a protein O-mannosyltransferase, contributes to virulence through governing the appressorium turgor pressure in Metarhizium acridum

O-glycosylation is a very important post-translational modification of protein and involved in many cell processes in fungi. There exist three protein O-manosyltransferanse genes (MaPmt1, MaPmt2, MaPmt4) in Metarhizium acridum based on sequence homology. Here, MaPmt1, a gene for Pmt1 O-manosyltransferanse in M. acridum, was characterized and functionally analyzed through targeted gene disruption and complementation methods. Deletion of MaPmt1 had no effect on conidial germination, but slightly increased the conidial yield and significantly impaired fungal tolerances to UV-B radiation and wet-heat. Deletion of MaPmt1 made the fungus become more sensitive to cell wall disturbing agents and exhibit a thinner cell wall with changed components. Insect bioassays showed that disruption of MaPmt1 attenuated the fungal virulence significantly by topical inoculation but not by injection, indicating that MaPmt1 is required for penetration during the infection of M. acridum. Interestingly, deletion of MaPmt1 did not affect appressorium formation but significantly decreased appressorium turgor pressure. Moreover, the decreased virulence of MaPmt1 disruptant is mainly due to the reduced appressorium turgor pressure, which may be resulted from the declined glycerol concentration, combined with the weakened cell wall that could not hold the normal appressorium turgor pressure to penetrate the host cuticle.

Immunotranscriptome analysis of Plutella xylostella reveals differences in innate immune responses to low- and high-virulence Beauveria bassiana strain challenges.

Entomopathogenic fungi have developed multiple strategies to overcome the immune defenses of their target insects, whereas insect pests have devised various defense mechanisms to combat fungal infection. However, differences in the molecular mechanisms of the innate immune defense strategies of insects upon infection with different fungal strains from the same species have not been reported. Two Beauveria bassiana strains were obtained that significantly varied in their pathogenicity but were comparable in terms of growth, conidial yield, and cuticle penetration. To investigate the molecular mechanisms underlying the immune response of Plutella xylostella infected with these two strains, RNA-Seq was performed 48 h after infection. A total of 1027 differentially expressed genes (DEGs) were identified, and more than 200 DEGs were enriched in Kyoto Encyclopedia of Genes and Genome (KEGG) pathways involved in disease response, revealing differences in the immune response of P. xylostella to different B. bassiana infections at 48 h. Twenty-eight of the DEGs were related to innate immune functions, such as pathogen recognition, immune system activation and antimicrobial reactions. RNA interference (RNAi)-mediated gene silencing assays showed that PxApoLIII and PxCSP played critical roles in the P. xylostella immune response. PxApoLIII was expressed at higher levels during infection with the high-virulence strain, whereas PxCSP showed the opposite expression pattern during infection with the low-virulence strain, indicating that PxApoLIII and PxCSP might participate in P. xylostella innate immune defense against high- and low-virulence B. bassiana strains. The present findings demonstrate that strains of a single species of pathogenic fungi that differ in virulence can induce the expression of different genes in P. xylostella. These results advance our knowledge of the molecular mechanisms underlying fungi-pest interactions.

The secondary metabolite regulator, BbSmr1, is a central regulator of conidiation via the BrlA‐AbaA‐WetA pathway in Beauveria bassiana

The filamentous fungus Beauveria bassiana, an insect fungal pathogen, is widely used for pest biocontrol. Aerial conidia are infectious propagules, and their yield and viability greatly affect the field application of this fungus; however, little is known about the molecular regulatory mechanism of the triggered conidiation. In the present study, we find that the secondary metabolite regulator BbSmr1 is involved in the regulation of asexual conidiation development and stress response in B. bassiana. A deficiency in Bbsmr1 results in a prominent fluffy-like phenotype on solid medium, decreased conidial yield, accelerated conidial germination, as well as increased tolerance to H2 O2 stress and cell wall inhibitors. The deletion of Bbsmr1 also leads to thickened conidial cell walls and changed cell epitopes. Overexpressing either BbbrlA or BbabaA in the ∆Bbsmr1 strain can rescue the phenotypes of conidial development and stress response. BbSmr1 activates BbbrlA transcription by directly binding to the A4GA3 sequence of the BbbrlA promoter. BbBrlA in turn binds to the promoter of Bbsmr1 and negatively regulates the expression of Bbsmr1. These results indicate that BbSmr1 positively regulates conidial development in B. bassiana by activating the central development pathway BrlA-AbaA-WetA and provides insights into the developmental regulatory mechanism of entomopathogenic fungi.

Selection of Beauveria bassiana (Hypocreales: Cordycipitaceae) strains to control Xyleborus affinis (Curculionidae: Scolytinae) females.

BackgroundXyleborus affinis Eichhoff (Coleoptera: Curculionidae) is an ambrosia beetle reported to affect avocado trees (Persea americana Mill.). The use of the entomopathogenic fungus (EPF) Beauveria bassiana (Bals.-Criv.) Vuill. for ambrosia beetle control represents an alternative to insecticides.MethodsThis study was designed in two stages to select B. bassiana strains with potential to control X. affinis females. In the first stage, 19 B. bassiana Mexican strains from EPF collection, isolated from Coleoptera (CHE-CNRCB, http://www.gob.mx/senasica/documentos/coleccion-de-hongos-entomopatogenos), were tested. Analyses included radial growth rate, conidial yield, spore germination, and germ tube length. Results were analysed by Principal Component Analysis (PCA) to identify clusters within favourable growth phenotypes. For the second stage, 10 selected strains were re-analysed for virulence-related metabolic characteristic, including cell wall-bound cuticle-degrading enzymes–Pr1-like proteases and β-N-acetyl glucosaminidases (NAGase) chitinases, conidial hydrophobicity and monopolar germination parameters. A second PCA analysis was run for those virulence parameters analysed, and upon results strains CHE-CNRCB 44, 171, 431 and 485 were selected and tested against X. affinis females. Females were treated with a 1 × 108 conidia mL−1 suspension (recommended rate), using a Potter Tower.ResultsAll strains showed insecticidal activity, inducing up to 58% mortality; about 30% dead beetles developed aerial mycelia (CHE-CNRCB 485) and the fastest mortality rate was t0 = 1.95 (CHE-CNRCB 44).ConclusionSince all selected strains showed virulence against X. affinis females, results indicated the possibility of selecting B. bassiana strains based on multiple metabolic attributes, as a preliminary test to perform bioassays against order-related target insects.

Colonization of Beauveria bassiana 08F04 in root-zone soil and its biocontrol of cereal cyst nematode (Heterodera filipjevi).

Cereal cyst nematodes cause serious yield losses of wheat in Hunaghuai winter wheat growing region in China. Beauveria bassiana 08F04 isolated from the surface of cysts is a promising biological control agent for cereal cyst nematodes. As the colonization capacity is a crucial criteria to assess biocontrol effectiveness for a microbial agent candidate, we aimed to label B. bassiana 08F04 for efficient monitoring of colonization in the soil. The binary pCAM-gfp plasmid containing sgfp and hph was integrated into B. bassiana 08F04 using the Agrobacterium tumefaciens-mediated transformation. The transformation caused a significant change in mycelial and conidial yields, and in extracellular chitinase activity in some transformants. The cultural filtrates of some transformants also decreased acetylcholinesterase activity and the survival of Heterodera filipjevi second-stage juveniles relative to the wild-type strain. One transformant (G10) had a growth rate and biocontrol efficacy similar to the wild-type strain, so it was used for a pilot study of B. bassiana colonization conducted over 13 weeks. Real-time PCR results and CFU counts revealed that the population of G10 increased quickly over the first 3 weeks, then decreased slowly over the following 4 weeks before stabilizing. In addition, the application of wild-type B. bassiana 08F04 and transformant G10 significantly reduced the number of H. filipjevi females in roots by 64.4% and 60.2%, respectively. The results of this study have practical applications for ecological, biological and functional studies of B. bassiana 08F04 and for bionematicide registration.

Elevated air temperature shifts the interactions between plants and endophytic fungal entomopathogens in an agroecosystem

Abstract The possible effects of climate change on species interactions remain a very complex and challenging subject in community ecology. Here, we comparatively examine the interactions between maize (Zea mays) and an endophytic fungal entomopathogen (Beauveria bassiana) in a typical agroecosystem under both ambient and elevated air temperatures. We found that under ambient temperature certain key biological characteristics in maize (i.e., height, relative growth rate, biomass, and defense enzymes) and B. bassiana (i.e., conidia yield, germination rate of conidia, and virulence) were positively related to each other. Under elevated air temperature, however, we only detected positive effects of maize on B. bassiana (i.e., conidia yield, germination rate of conidia, and virulence), but little effect of B. bassiana on maize. These observations suggest that elevated air temperature could shift the interactions between plants and the endophytic fungal entomopathogens, possibly even from mutualism to commensalism. Both the nature and strength of species interactions are important for better understanding and predicting structure and stability of ecological communities in agroecosystems under climate change.

Genome-wide transcriptomic analysis of the response of Botrytis cinerea to wuyiencin.

Grey mould is caused by the ascomycetes Botrytis cinerea in a range of crop hosts. As a biological control agent, the nucleoside antibiotic wuyiencin has been industrially produced and widely used as an effective fungicide. To elucidate the effects of wuyiencin on the transcriptional regulation in B. cinerea, we, for the first time, report a genome-wide transcriptomic analysis of B. cinerea treated with wuyiencin. 2067 genes were differentially expressed, of them, 886 and 1181 genes were significantly upregulated and downregulated, respectively. Functional categorization indicated that transcript levels of genes involved in amino acid metabolism and those encoding putative secreted proteins were altered in response to wuyiencin treatment. Moreover, the expression of genes involved in protein synthesis and energy metabolism (oxidative phosphorylation) and of those encoding ATP-binding cassette transporters was markedly upregulated, whereas that of genes participating in DNA replication, cell cycle, and stress response was downregulated. Furthermore, wuyiencin resulted in mycelial malformation and negatively influenced cell growth rate and conidial yield in B. cinerea. Our results suggest that this nucleoside antibiotic regulates all aspects of cell growth and differentiation in B. cinerea. To summarize, some new candidate pathways and target genes that may related to the protective and antagonistic mechanisms in B. cinerea were identified underlying the action of biological control agents.