Toxicity of crude toxin protein produced by Cordyceps fumosorosea IF-1106 against Myzus persicae (Sulze).

The entomopathogenic fungus Cordyceps fumosorosea IF-1106 is a potential biocontrol agent with high pathogenicity to the aphid Myzus persicae. We extracted the crude toxin protein from a liquid culture broth of an isolated C. fumosorosea strain using the ammonium sulfate precipitation method, and its toxicity to Myzus persicae was measured by injection, oral exposure, and topical exposure. The crude toxin protein of C. fumosorosea had insecticidal activity against M. persicae. Body cavity injection and oral exposure had significantly higher insecticidal activity against adults than contact sprays. The highest cumulative corrected mortality of adults after injection was 81.85 ± 13.45 %, and the highest cumulative corrected mortality of adults after ingestion was 85.45 ± 11.88 %. The proportion of plasmatocytes in adult blood lymphocytes reached the highest at 3 days after injection and feeding, and the proportion of granulocytes was the highest at 2 days after injection and feeding. These data confirmed the toxicity of the crude toxin protein of C. fumosorosea toxin to M. persicae and helped clarify the pathogenic mechanism of the strain. Population management of M. persicae may be possible by using a natural toxic compound produced by C. fumosorosea that is selective to this pest species.

Development of a fluid-bed coating process for soil-granule-based formulations of Metarhizium brunneum, Cordyceps fumosorosea or Beauveria bassiana.

AIM: Granule-based products of solid state fermented micro-organisms are available for biocontrol. Because liquid fermentation has several advantages, we investigated fluid-bed coating with liquid fermented biomass. METHODS AND RESULTS: Biomass containing mycelium or mycelium and submerged spores of the entomopathogenic fungi Metarhizium brunneum, Cordyceps fumosorosea and Beauveria bassiana were produced in liquid culture, separated and different biomass concentrations were adjusted. Based on the examined thermo-tolerance, we defined fluid-bed coating adjustments and investigated granule colonization and sporulation on granules. Granule colonization depended on the biomass concentration and strain. For C. fumosorosea and B. bassiana, concentrations of 0·003%dry weight resulted in nearly 100% granule colonization, for M. brunneum with concentrations of 0·7%dry weight in only 50%. The conidiation on granules in sterile soil was highly influenced by the moisture content. Because the granule colonization of M. brunneum was unsatisfactory, we pre-coated nutrients followed by coating with biomass, submerged spores or conidia. Malt extract had a positive effect on the granule colonization for biomass and submerged spores. Furthermore, aerial conidia can also be coated. CONCLUSIONS: Fluid-bed coating of fungal biomass is suitable for the development of granules. SIGNIFICANCE AND IMPACT OF THIS STUDY: With this technology, cost-efficient biocontrol products can be developed.

The conserved role of miR-2 and novel miR-109 in the increase in fecundity of Diaphorina citri induced by symbiotic bacteria and pathogenic fungi.

Infection with pathogens can increase the fecundity and other fitness-related traits of insect vectors for their own advantage. Our previous research has reported the pivotal role of DcKr-h1 in the fecundity improvement of Diaphorina citri induced by the bacterium, "Candidatus Liberibacter asiaticus" (CLas), and the fungus, Cordyceps fumosorosea (Cf). However, the posttranscriptional regulation of this process remains poorly understood. Given the significance of miRNAs in gene regulation, we delved into their roles in shaping phenotypes and their underlying molecular mechanisms. Our results indicated that two miRNAs, miR-2 and novel-miR-109, jointly inhibited DcKr-h1 expression by binding to its 3' untranslated region (UTR). In the D. citri-CLas interaction, the expression levels of miR-2 and novel-miR-109 in the ovaries of CLas-positive psyllids were lower compared to CLas-negative individuals. Overexpression of miR-2 or novel-miR-109 significantly decreased fecundity and CLas titer in ovaries and caused reproductive defects reminiscent of DcKr-h1 knockdown. Similarly, in the D. citri-Cf interaction, the levels of miR-2 and novel-miR-109 markedly decreased in the ovaries. Upregulation of miR-2 or novel-miR-109 also resulted in reduced fecundity and ovary defects similar to those caused by DcKr-h1 silencing. Moreover, feeding antagomir-2 or antagomir-109 partially rescued the defective phenotypes caused by DcKr-h1 silencing in both model systems, and miR-2 and novel-miR-109 were repressed by juvenile hormone (JH) and regulated the genes associated with egg development. This study shows a conserved regulatory mechanism, whereby JH suppresses the expression of miR-2 and novel-miR-109 which, together with JH-induced transcription of DcKr-h1, increases female fecundity induced by both symbiotic bacteria and pathogenic fungi. IMPORTANCE: Infection with pathogens can increase the fecundity and other fitness-related traits of insect vectors for their own advantage. Our previous research has reported that DcKr-h1 plays a critical role in the increase in fecundity of Diaphorina citri induced by the bacterium, "Candidatus Liberibacter asiaticus" (CLas) and the fungus, Cordyceps fumosorosea (Cf). However, the posttranscriptional regulation of this process remains poorly understood. Given the significance of miRNAs in gene regulation, we delved into their roles in shaping phenotypes and their underlying molecular mechanisms. Our results indicated that two miRNAs, miR-2 and novel-miR-109, jointly inhibited DcKr-h1 expression by binding to its 3' untranslated region (UTR). In both D. citri-CLas and D. citri-Cf interactions, the increased juvenile hormone (JH) titer and reduced abundance of miR-2 and novel-miR-109 ensure high levels of DcKr-h1 expression, consequently stimulating ovarian development and enhancing fecundity. These observations provide evidence that miR-2 and miR-109 are crucial players in the JH-dependent increase in fecundity in psyllids induced by infection with different pathogens.

Effect of Abiotic Factors on Fumosorinone Production from Cordyceps fumosorosea via Solid-State Fermentation.

Cordyceps fumosorosea is an important species in the genus of Cordyceps, containing a variety of bioactive compounds, including fumosorinone (FU). This study was a ground-breaking assessment of FU levels in liquid and solid cultures. The present study focused on the impacts of solid-state fermentation (SSF) using solid substrates (wheat, oat, and rice), as well as the effects of fermentation parameters (pH, temperature, and incubation period), on the generation of FU. All the fermentation parameters had significant effects on the synthesis of FU. In a study of 25 °C, 5.5 pH, and 21 days of incubation period combinations calculated -to give maximal FU production, it was found that the optimal values were 25 °C, 5.5 pH, and 21 days, respectively. In a solid substrate medium culture, FU could be produced from SSF. At 30 days, a medium composed of rice yielded the most FU (798.50 mg/L), followed by a medium composed of wheat and oats (640.50 and 450.50 mg/L), respectively. An efficient method for increasing FU production on a large scale could be found in this approach. The results of this study might have multiple applications in different industrial fermentation processes.

Effectiveness of Cordyceps fumosorosea Wettable Powder Formulation against Metisa plana (Walker) and Its Side Effects on Elaeidobius kamerunicus in Oil palm Plantation.

Development of mycoinsecticides with Cordyceps fumosorosea as an active ingredient is established as an alternate way to control the Metisa plana population while reducing chemical insecticide dependence. Three mycoinsecticide formulations (SS6, SS7, and SS8) with dispersing and wetting agents were developed as wettable powder formulations in this trial. SS8 demonstrated the best wettability, suspensibility, and dispersibility with viability at 107 (CFU)/mL even after three months of storage. However, SS7 developed with C. fumosorosea as an active ingredient was found to effectively reduce the bagworm population by more than 95%. The application of all mycoinsecticide formulations in the infested oil palm area was able to reduce the M. plana population by more than 95%, 30 DAT. The formulations also show no significant increase in mortality of the oil palm pollinator, Elaeidobius kamerunicus. This finding indicates that the C. fumosorosea tested has potential for managing bagworms without harming pollinators on oil palm plantations.

Fungal strain and crop cultivar affect growth of sweet pepper plants after root inoculation with entomopathogenic fungi.

As endophytes, entomopathogenic fungi can protect plants against biotic and abiotic stresses and at the same time promote plant growth and plant health. To date, most studies have investigated whether Beauveria bassiana can enhance plant growth and plant health, while only little is known about other entomopathogenic fungi. In this study, we evaluated whether root inoculation of the entomopathogenic fungi Akanthomyces muscarius ARSEF 5128, B. bassiana ARSEF 3097 and Cordyceps fumosorosea ARSEF 3682 can promote plant growth of sweet pepper (Capsicum annuum L.), and whether effects are cultivar-dependent. Plant height, stem diameter, number of leaves, canopy area, and plant weight were assessed four weeks following inoculation in two independent experiments using two cultivars of sweet pepper (cv. 'IDS RZ F1' and cv. 'Maduro'). Results showed that the three entomopathogenic fungi were able to enhance plant growth, particularly canopy area and plant weight. Further, results showed that effects significantly depended on cultivar and fungal strain, with the strongest fungal effects obtained for cv. 'IDS RZ F1', especially when inoculated with C. fumosorosea. We conclude that inoculation of sweet pepper roots with entomopathogenic fungi can stimulate plant growth, but effects depend on fungal strain and crop cultivar.

Metabolites and novel compounds with anti-microbial or antiaging activities from Cordyceps fumosorosea.

High-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) analysis revealed that there are 20 main components in spores and mycelia extract of Cordyceps fumosorosea strain RCEF 6672 including mannitol (1), uridine (2), adenine (3). N6-(2-hydroxyethyl)-adenosine (4). N6-(2-hydroxyethylacetate)-adenosine (5), fumosoroseanoside A (6) and B (7), ovalicin-4α-alcohol (8), 1-linoleoyl-sn-glycero-3-phosphocholine (9) and its isomer (10), fumosoroseain A (11) and its isomer (12), 5 non-ribosomal peptides (13 to 17) and 3 fatty acids (18 to 20). The compounds 5, 6, 7, 9 and 11 were prepared with preparative and semi-preparative HPLC and identified with 1D and 2D NMR. Compounds 4 and 5 were the first time identified from C. fumosorosea. Compounds 6, 7 and 11 are novel compounds. Compounds 6 and 7 showed antibacterial and antifungal activities, and 11 showed antiaging activity. All the secondary metabolites (4 to 8 and 11 to 17) have strong bioactivities indicating that the metabolites have pharmaceutical development potentiality.

Control of Fusarium and nematodes by entomopathogenic fungi for organic production of Zingiber officinale.

Ginger (genus Zingiber) is widely used as a spice and a medicinal herb worldwide and is the major ingredient of traditional local drinks such as jamu in Southeast Asia. Because ginger is frequently consumed, there is an increasing interest in organic ginger production without the use of synthetic agrochemicals. Recent studies have reported that certain kinds of entomopathogenic fungi (EPF) can establish endophytic- or mycorrhiza-like relationships with plants, thereby promoting plant growth and health, in addition to their typical role in crop protection as biological control agents. In this study, we explored the possibility of non-entomopathogenic effects of EPF Beauveria bassiana and Cordyceps fumosorosea on ginger plants (Zingiber officinale) via antagonism with Fusarium oxysporum or the parasitic nematode Meloidogyne incognita. The two EPF negatively affected the growth of F. oxysporum and survival of M. incognita in vitro. The application of EPF did not have any negative effect on the growth of ginger plants. Soil chemical properties were not different between the plots with or without EPF application, while the diversity of soil bacteria was observed to increase on application of EPF. At least C. fumosorosea appeared to persist in soil during the period of ginger cultivation. Thus, these EPF are potentially useful tools for producing chemical-free ginger.

Characterization and Toxicity of Crude Toxins Produced by Cordyceps fumosorosea against Bemisia tabaci (Gennadius) and Aphis craccivora (Koch).

Cordyceps fumosorosea, an insect pathogenic fungus, produces different toxins/secondary metabolites which can act as pest control agents. This study reports the extraction and characterization of crude mycelial extracts of C. fumosorosea isolate SP502 along with their bio-efficacy against Bemisia tabaci and Aphis craccivora. Fourier transform infrared spectroscopy, liquid chromatography, mass spectrometery and nuclear magnetic resonance analysis of C. fumosorosea isolate SP502 extracts showed the presence of five major compounds-Trichodermin, 5-Methylmellein, Brevianamide F, Enniatin and Beauvericin-which all may potentially be involved in insecticidal activity. The HPLC analysis of C. fumosorosea mycelial extracts and Beauvericin standard showed similar chromatographic peaks, with the content of Beauvericin in the crude toxin being calculated as 0.66 mg/ml. The median lethal concentrations of C. fumosorosea mycelial extracts towards first, second, third and fourth instar nymphs of A. craccivora were 46.35, 54.55, 68.94, and 81.92 µg/mL, respectively. The median lethal concentrations of C. fumosorosea mycelial extracts towards first, second, third and fourth instar nymphs of B. tabaci were 62.67, 72.84, 77.40, and 94.40 µg/mL, respectively. Our results demonstrate that bioactive compounds produced by C. fumosorosea isolate SP502 have insecticidal properties and could, therefore, be developed into biopesticides for the management of B. tabaci and A. craccivora.

Synthesis of Cordyceps fumosorosea-Biochar Nanoparticles and Their Effects on Growth and Survival of Bemisia tabaci (Gennadius).

Nanotechnology can offer an environmentally sustainable alternative to synthetic chemicals for pest management. Nano-formulations of different microbial pest control agents have been effective against several insect pests. Synthesis of Cordyceps fumosorosea-biochar (BC) nanoparticles and their bio-efficacy against Bemisia tabaci was observed during this study. The characterization of C. fumosorosea-BC nanoparticles through different analytical techniques showed successful synthesis of nanoparticles. UV spectroscopy showed a characteristic band of surface plasmon between 350 and 400 nm; SEM images confirmed the synthesis of spherical shaped nanoparticles; X-ray diffractogram showed strong peaks between 2θ values of 20°-25°; and atomic force microscopy (AFM) analysis revealed particle size of 49.151 nm. The bioassay studies demonstrated that different concentrations of C. fumosorosea-BC nanoparticles caused significant reduction in hatchability of B. tabaci eggs as well as survival of immatures emerging from treated eggs when compared with controls. The results also revealed that C. fumosorosea-BC nanoparticles were highly pathogenic against 2nd and 3rd instar nymphs and pupae of B. tabaci having LC50 values of 6.80, 7.45, and 8.64 ppm, respectively. The LT50 values for 20 ppm concentration of C. fumosorosea-BC nanoparticles against 2nd and 3rd instar nymphs, and pupae of B. tabaci were 3.25 ± 0.29, 3.69 ± 0.52, and 4.07 ± 0.51 days, respectively. These findings suggest that C. fumosorosea-BC nanoparticles can potentially be used in biorational B. tabaci management programs.

Unraveling the Mode of Action of Cordyceps fumosorosea: Potential Biocontrol Agent against Plutella xylostella (Lepidoptera: Plutellidae).

The entomopathogenic fungus, Cordyceps fumosorosea is a potential eco-friendly biocontrol agent. The present study revealed the entire course of infection of P. xylostella by C. fumosorosea with particular reference to cuticular penetration. Comparative studies on the infection of Plutella xylostella larvae by two strains of C. fumosorosea with different pathogenicity were carried out using light, scanning, and transmission electron microscopy. We found that C. fumosorosea tended to adhere to the cuticle surfaces containing protrusions. Although conidia of the lower pathogenic strain IFCF-D58 germinated, they failed to penetrate and complete the development cycle. In contrast, the higher pathogenic strain IFCF01 began to germinate within 4 h and attached to the cuticle by a thin mucilaginous matrix within 8 h post-inoculation. After 24 h post-inoculation, germ tubes and penetrating hyphae reached the cuticular epidermis and began to enter the haemocoel. Within 36 h post-inoculation, the hyphal bodies colonized the body cavity. Hyphae penetrated from inside to outside of the body after 48 h and sporulated the cadavers. After 72 h post-inoculation, numerous conidia emerged and the mycelial covered the entire cuticular surface. The two strains showed similarities in terms of conidial size and germination rate. However, IFCF-D58 exhibited significantly fewer appressoria and longer penetrating hyphae compared to the more infective IFCF01 on all surface topographies. The current pathogen invasion sequence of events suggested that the aggressive growth and propagation along with rapid and massive in vivo production of blastospores facilitate the conidia of IFCF01 to quickly overcome the diamondback moth's defense mechanism.

In Planta Localization of Endophytic Cordyceps fumosorosea in Carrizo Citrus.

Entomopathogenic fungi can be a useful resource for controlling insect vectors of citrus plant pathogens, such as the Asian citrus psyllid (Diaphorina citri) associated with huanglongbing or the citrus root weevil (Diaprepes abbreviatus) associated with the spread of Phytophtora spp. In this study, Cordyceps fumosorosea (Cfr) was investigated in planta as a potential endophytic entomopathogenic fungus and various inoculation techniques were used to determine if it would colonize the Carrizo citrange (Citrus × insitorum) seeds and plants. The four inoculation methodologies evaluated were seed soaking, stem injection, foliar spray, and soil drench. Seed immersion trials demonstrated that the roots of the Carrizo citrange plant can be inoculated successfully with Cfr. Stem injection, foliar spray, and soil drench also provided successful inoculation of Cfr. However, this fungus was only endophytic in the plant stem. Sand cores indicated that Cfr moved down through the sand column and was able to inoculate the roots. Given the prevalence of Cfr in the soil during the drench experiment, and that the fungus was able to colonize Carrizo citrange roots through seed immersion, this finding provides evidence of the potential endophytism of this fungus when applied to citrus plant species.

In Vitro Effects of Leaf Extracts from Brassica rapa on the Growth of Two Entomopathogenic Fungi.

This study aimed to determine the inhibitive or stimulatory effects of leaf extracts from two Brassica rapa subspecies on the hyphal growth of two well-known entomopathogenic fungi, Cordyceps fumosorosea and Beauveria bassiana. Extract concentrations of 50, 25, and 10% w/v based on leaf fresh weight were prepared from turnip (B. rapa subspecies rapa) and bok choy (B. rapa subspecies chinensis) leaves. Each concentration was individually incorporated into potato dextrose agar plates for in vitro bioassays. The center of each plate was inoculated with 20 µL of a fungal suspension that was allowed 24 h to soak into the agar before sealing the plates and incubating them at 25 °C under a 14-h photophase. The fungal colony perimeter was marked 5 days after inoculation on two perpendicular lines drawn on the bottom of each plate. Radial colony growth was measured from 4 marks per plate 5, 10, and 15 days later. Radial growth rates for both fungi were 1.3-2.0 and 0.9-1.4 times faster with bok choy and turnip extracts, respectively, at the 25% and 50% concentrations compared to the no-extract control treatment. Therefore, bok choy and turnip leaf extracts can stimulate entomopathogenic fungus growth within 15 days. Biochemical compounds in the extracts include sesquiterpenes, α-copaene, ß-selinene, γ-gurjunene, calamenene, cubenene, and α-calacorene.

Host-dependent contributions of the Cfcdp1 protease gene to virulence in the entomopathogenic fungus Cordyceps fumosorosea.

BACKGROUND: The genomes of broad host range insect pathogenic fungi, including Cordyceps fumosorosea, encode for a suite of secreted proteases implicated in targeting, penetration, and degradation of the host exoskeleton or cuticle. These cuticle-degrading proteases act as critical virulence factors, but their functions within the biological context, particularly in relation to host specificity, remain poorly characterized. RESULT: A C. fumosorosea protease gene, Cfcdp1, was identified and a targeted gene-knockout strain constructed. Minor growth defects were observed for the ΔCfcdp1 strain when compared to the wild-type parent and complemented (ΔCfcdp1::Cfcdp1) strains, with delayed and decreased sporulation noted for the mutant. Decreased subtilisin-like protease activity was seen for the ΔCfcdp1 strain, although total secreted protease activity was similar between the mutant and wild-type strains. Insect bioassays using whitefly, Bemisia tabaci, and cabbageworm, Pieris rapae, showed decreased infectivity, i.e. 2.4-3.4-fold increase in lethal dose (LC50 ) and an increased time to death (LT50 ), for the ΔCfcdp1 strain. In contrast, insect bioassays using the diamondback moth, Plutella xylostella, or the brown planthopper, Nilaparvata lugens, showed increased infectivity, i.e. a 3-5-fold decrease in LC50 , and a decreased LT50 . Differential effects were also seen on the fecundity of B. tabaci infected by the different fungal strains. CONCLUSION: These data reveal host-dependent effects of a protease implicated in cuticle degradation on C. fumosorosea virulence. The implications of these findings in suggesting context-dependent requirements of cuticle-degrading enzymes and their potentially differential roles in mediating virulence towards different hosts are discussed. © 2019 Society of Chemical Industry.

Endophytic Isolates of Cordyceps fumosorosea to Enhance the Growth of Solanum melongena and Reduce the Survival of Whitefly (Bemisia tabaci).

This study reports the effects of seed treatment with Cordyceps fumosorosea on seed germination, growth, colonization of eggplant (Solanum melongena), and growth of Bemisia tabaci (feeding on fungal colonized eggplant leaves). Germination rates of eggplant seeds were similar among different treatments. The growth parameters such as root length, shoot length, and number of leaves) differed significantly after 15, 30, and 60 days of seed treatment. The total dry weight of eggplant in response to treatment with C. fumosorosea isolates increased significantly when compared with the control. Both isolates of C. fumosorosea colonized different plant tissues, although the extent of colonization decreased during the experimental period. The colonization of eggplants by both C. fumosorosea isolates resulted in a significant reduction of B. tabaci incidence. This study possibly provides the first report of increased plant growth and increased insect mortality in eggplants inoculated with C. fumosorosea isolates.