Mass production of Beauveria bassiana for regulation of Leptinotarsa decemlineata populations

A simple liquid medium which enhanced the production of conidiospores by an isolate of the entomophagous fungus Beauveria bassiana is described. Spore production was attained using cultures floating in inflated sections of plastic tubing (“polyethylene cushions”) and glass bottles. A method is described for determining lethal doses and lethal times for second- and third-instar Leptinotarsa decemlineata larvae.     

Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions

Natural infection of Galleria mellonella larvae with the entomopathogenic fungus Beauveria bassiana led to antifungal, but not antibacterial host response. This was manifested by induction of gallerimycin and galiomicin gene expression and, consequently, the appearance of antifungal activity in the hemolymph of the infected larvae. The activity of lysozyme increased at the beginning of infection and dropped while infection progressed. Exposure of the naturally infected animals to 43 °C for 15 min extended their life time.Galleria mellonella larvae were injected with 10⁴, 10⁵ and 10⁶ fungal blastospores, resulting in the appearance of strong antifungal activity and a significant increase in lysozyme activity in larval hemolymph after 24 h. Antibacterial activity was detectable only when 10⁵ and increased when 10⁶ blastospores were injected. The number of the injected B. bassiana blastospores also determined the survival rate of animals. We found that exposure of the larvae to 38 °C for 30 min before infection extended their life time when 10³ and 10⁴ spores were injected. The increase in the survival rate of the pre-heat-shocked animals may be explained by higher expression of antimicrobial peptides and higher antifungal and lysozyme activities in their hemolymph in comparison to non-heat-shocked animals.     

Chitinase is an inducible enzyme in Beauveria bassiana

Sterilization of chitin by autoclaving or boiling causes release of d-glucosamine and N-acetylglucosamine from the macromolecule and these solubilized components actually function as the inducers for synthesis of chitinase. The insoluble macromolecule is not an inducer of chitinase since sterilization by dry heat or chloroform will not bring about release of the amino sugars or induction of the enzyme. Free glucosamine, N-acetylglucosamine, and chitobiose are all good inducers of chitinase. Most sustained synthesis of the enzyme occurs in an autoclaved chitin-salts medium.     

Insect-toxic secreted proteins and virulence of the entomopathogenic fungus Beauveria bassiana

Fungal virulence has been mostly associated with cuticle-degrading enzymes that can be regulated depending on nutrient conditions. However, few studies have related fungal virulence to insect-toxic secreted proteins. Here, we describe how the presence of secreted toxic proteins may be linked to conidial virulence, which can be affected by nutrient factors. In this study we evaluated: (1) the virulence of the conidia of four Beauveria bassiana strains (EABb 01/103-Su, EABb 01/12-Su, EABb 01/88-Su and EABb 01/110-Su) grown on three different media (malt extract agar (MA), Rice (Rice), Sabouraud dextrose agar (SDA) and harvested from the cadavers of fungal-infected Galleria mellonella larvae (CAD) and (2) the toxicity of the crude soluble protein extracts (CSPEs) obtained from Adamek’s liquid medium inoculated with these conidia. Conidial suspensions were obtained from the four media, assessed on G. mellonella larvae and used to produce CSPEs that were injected into healthy G. mellonella larvae. The larvae were also injected with conidia obtained from MA and CAD cultures to expose them to in vivo-secreted proteins. For all isolates, the CAD conidia were by far the most virulent, followed by conidia grown on SDA, Rice and MA. The injected CSPEs showed the same toxicity trends as the conidial suspensions. In addition, the outcomes of injection of the in vivo-secreted proteins showed that the toxic proteins secreted in vitro by the EABb 01/110-Su strain are not produced in vivo. However, the other strains produced toxic proteins both in vivo and in vitro, suggesting that these toxic proteins may be virulence factors involved in invertebrate pathogenesis.     

Nutritional requirements for conidial germination and hyphal growth of Beauveria bassiana

Nutritional requirements for germination and growth of the entomopathogenic fungus Beauveria bassiana are not complex. For germination to occur, a utilizable source of carbon must be present; however, a nitrogen source is needed for continued hyphal growth, otherwise lysis ensues. Compounds that can serve as utilizable carbon-energy sources for germination include glucose, N-acetylglucosamine, glucosamine, chitin, starch, lanolin, hydrocarbons in crude oil, and some longer-chain fatty acids. Both organic and inorganic sources of nitrogen are readily utilized for growth. Conidia undergo active metabolism soon after being placed in a suitable growth medium, indicating that conidia are released from their state of dormancy several hours before emergence of the germ tube can be observed. Because of the nutritional versatility of B. bassiana, this fungus should be able to survive and be infective in several types of natural environments.     

Utilizable surface nutrients on Heliothis zea available for growth of Beauveria bassiana

Amino acids and glucosamine are present on the surface of Heliothis zea larvae. The amino acid compositions vary among instars and with time following completion of the gross molting process. Larvae collected from ears of corn have many surface amino acids. All amino acid combinations found on larval surfaces are sufficient for initiation of germination and growth by Beauveria bassiana. Amines and peptides are also present; these do not inhibit germination of B. bassiana or Aspergillus niger.     

Culture degeneration in conidia of Beauveria bassiana and virulence determinants by proteomics

The quality of Beauveria bassiana conidia directly affects the virulence against insects. In this study, continuous subculturing of B. bassiana on both rice grains and potato dextrose agar (PDA) resulted in 55 and 49 % conidial yield reduction after 12 passages and 68 and 60 % virulence reduction after 20 and 12 passages at four d post-inoculation, respectively. The passage through Tenebrio molitor and Spodoptera exigua restored the virulence of rice and PDA subcultures, respectively. To explore the molecular mechanisms underlying the conidial quality and the decline of virulence after multiple subculturing, we investigated the conidial proteomic changes. Successive subculturing markedly increased the protein levels in oxidative stress response, autophagy, amino acid homeostasis, and apoptosis, but decreased the protein levels in DNA repair, ribosome biogenesis, energy metabolism, and virulence. The nitro blue tetrazolium assay verified that the late subculture's colony and conidia had a higher oxidative stress level than the early subculture. A 2A-type protein phosphatase and a Pleckstrin homology domain protein Slm1, effector proteins of the target of rapamycin (TOR) complex 1 and 2, respectively, were dramatically increased in the late subculture. These results suggest that TOR signalling might be associated with ageing in B. bassiana late subculture, in turn affecting its physiological characteristics and virulence.     

Effects of sheep fleece washings on the germination and growth of Beauveria bassiana

The development of biopesticides against ectoparasites must take into account the effect that an animal host’s secretions and host associated micro-organisms may have on the viability of the applied agent. In this study, the effects of secretions washed from the pelt of sheep on the viability and growth of the fungal pathogen Beauveria bassiana, were assessed. The fungal isolate had been obtained from the parasitic sheep scab mite Psoroptes ovis. It was added to 0.05% Tween 80 in which sheep fleece had been washed up to six times, to ascertain whether successive washings had any effect on the viability of conidia over 6 days. The effects of sterile and non-sterile washings on viability and growth were also investigated. Results indicated that substances in the sheep fleece may cause a significant reduction in the viability of conidia. Viability was linked to the number of times the sheep pelt had been washed in the Tween, with conidia incubated in the first wash from the sheep pelt showing a significantly greater decrease in viability compared to those incubated in the sixth wash. Viability was not linked to the sterility of the washes, although there was a significant difference between length of germ tube growth from viable conidia in sterile and non-sterile washings.     

白僵菌施加对水稻三种抗氧化酶活力及叶际微生物多样性的影响

为了考察白僵菌使用后的生态安全性,研究了白僵菌施加对水稻3种保护酶活力及叶际微生物多样性的影响。使用荧光定量PCR对喷洒白僵菌孢子悬液的水稻叶际提取总DNA并进行荧光定量PCR扩增,发现白僵菌可以在水稻叶际残留达30 d之久,并且初始喷施浓度越大,衰减速率越高。与施加化学农药相比,施加白僵菌没有对水稻叶片的3种抗氧化酶活力带来不利影响。白僵菌处理组SOD、POD活力在第10天时高于对照组20.38%、8.65%,而CAT活力在第30天时最高高于对照组33.67%,在第30天时,化学农药导致CAT活力相比较对照组下降了42.71%。使用DGGE分析表明白僵菌对水稻叶际细菌、真菌微生物群落结构影响较小,并且白僵菌处理组微生物群落结构相似度、香农指数及条带数均较高。结果表明白僵菌是一种环境友好型的微生物农药。     

Comparative virulence of Beauveria bassiana isolates against lepidopteran pests of vegetable crops

Forty-three isolates of the entomopathogenic fungus Beauveria bassiana were screened for virulence against second-instar larvae of diamondback moth (Plutella xylostella) (DBM), European corn borer (Ostrinia nubilalis) (ECB), corn earworm (Helicoverpa zea) (CEW), and fall armyworm (Spodoptera frugiperda) (FAW); 30 of these isolates were tested against beet armyworm (Spodoptera exigua) (BAW). Highly virulent isolates were also tested against black cutworm (Agrotis ipsilon) (BCW), and the most virulent isolate was also assayed against imported cabbage worm (Pieris rapae) (ICW) and cabbage looper (Trichoplusia ni) (CL). All lepidopteran species tested were susceptible to B. bassiana. Corn earworm and beet armyworm were most susceptible to fungal infection, and fall armyworm was least susceptible. Limited testing suggested low susceptibility of black cutworm and cabbage looper. B. bassiana isolate 1200 exhibited virulence against all pest species greater than or equal to commercial strain GHA of B. bassiana currently registered in the USA as BotaniGard®. In assays in which larvae were topically sprayed and maintained on the treated substrate for 24 h at 100% relative humidity, 6-day (25 °C) median lethal concentrations (LC₅₀s) of this isolate against CEW, BAW, DBM, FAW, ICW, ECB, CL, and BCW were 4, 5, 7, 11, 12, 98, 125, and 273 conidia/mm², respectively. The respective LC₅₀s of commercial strain GHA against these pest species were 9, 67, 97, 1213, 29, 1668, 541, and 3504 conidia/mm². Use of LC₅₀ versus median lethal concentration ratios (comparing LC₅₀s of each isolate to a “standard” strain) generated similar rankings of isolate virulence. Results from parametric ANOVAs of log LC₅₀ values followed by Tukey HSD multiple comparisons tests and those from Kruskal-Wallis nonparametric analyses followed by sequential Bonferroni tests for means comparisons were nearly identical.     

Mode of infection of the corn earworm (Heliothis zea) by Beauveria bassiana as revealed by scanning electron microscopy

Conidia from highly pathogenic mutants of Beauveria bassiana germinate quickly (within 18 hr) on the surface of corn earworm larvae (Heliothis zea) and immediately begin penetration of the cuticle. Enzymes produced by the penetrating hyphae degrade the cuticle since holes are formed at the point of entry. Clustering of conidia around nodules of larvae is often seen, but penetration is not restricted to such areas. Direct evidence is presented to show that conidia can also germinate inside of spiracle openings and could invade larvae by this route. Once inside the hemocoel, the fungus multiplies extensively; however, larval death occurs with only minimal breakdown of internal tissues. During mummification, outgrowths of fungal hyphae occur first and most extensively in the intersegmental regions of larvae. Conidia from mutants exhibiting low levels of pathogenicity are either significantly delayed or do not germinate on larval surfaces. When germination does occur, hyphae from such mutants do not penetrate immediately; instead, extensive surface hyphal growth, with or without eventual penetration of the integument, is evident.     

Age of three dipteran hosts as a factor governing the pathogenicity of Beauveria bassiana and Metarrhizium anisopliae

The age of three adult dipteran hosts was tested as a factor governing the virulence of two fungal pathogens. The subjects tested were 1-, 5-, 10-, 15-, 20-, 25-, and 30-day-old animals. The age of the house fly, Musca domestica, the age of the black blowfly, Phormia regina, and the age of the onion fly, Hylemya antiqua, do not significantly affect the virulence of either Beauveria bassiana or Metarrhizium anisopliae. B. bassiana kills M. domestica and P. regina more quickly than does M. anisopliae, but B. bassiana and M. anisopliae kill H. antiqua at the same rate. The ages of all three flies had no significant effect on average death time after inoculation with either pathogen.     

Relationship between virulence and repellency of entomopathogenic isolates of Metarhizium anisopliae and Beauveria bassiana to the termite Macrotermes michaelseni

Termites encounter a diverse array of potentially useful and harmful fungi in their subterranean habitats. These vary from symbiotic to harmful species with varying levels of virulence. How these hemiedaphic insects survive in habitats with infective fungi is not well understood. Possible mediation of olfactory signals in avoiding contact with entomopathogenic fungi has been explored by a number of workers. In the present study, we initially found that Macrotermes michaelseni detected a virulent isolate of Metarhizium anisopliae from some distance and avoided direct physical contact. We hypothesized that there may be a relationship between virulence and repellency of different isolates of M. anisopliae and Beauveria bassiana to the termite. We compared these for selected isolates of the two fungi. Positive correlations between the two parameters for both sets of isolates of the fungi were obtained. The results show an interesting co-evolutionary phenomenon in which the termite's response to either M. anisopliae or B. bassiana is directly related to potential harm these fungi can inflict on the insect and that the virulent strains are more likely to be recognized from some distance and avoided.     

In vivo events associated with entomopathology of Beauveria bassiana for the corn earworm (Heliothis zea)

When conidia of Beauveria bassiana are injected into the hemocoel of corn earworm larvae, it appears that a positive correlation exists between exocellular proteolytic activity of the fungus and entomopathological manifestations. Once inside the hemolymph, defense mechanisms (including phagocytosis) are incapable of overcoming the fungus and one important event in a terminal mycocidal cascade involves preferential invasion of the gut wall. Such invasion helps explain the observed inhibition of feeding by infected larvae. Although histopathological changes seen in gut tissues suggest that a gut toxin is produced, evidence for such a toxin could not be obtained in preliminary tests. Biochemical changes are seen in hemolymph components; however, these are viewed as being due to general starvation rather than to specific activities of the fungus, at least up to the time that a general mycosis is established. With the host larva under physiological stress (starvation, nutrient depletion, and, possibly, toxin production in gut tissues) and failure of defense mechanisms, the infection spreads quickly and a terminal mycosis results.     

Biodegradation of entomopathogenic hyphomycetes: Influence of clay coating on Beauveria bassiana blastospore survival in soil

The survival of naked and clay-coated Beauveria blastospores in soil was investigated with an experimental biodegradation method using a trap technique. At various times of incubation, traps were collected to study changes in biomass, inoculum potential, and infection potential of degraded blastospores, and to investigate the colonization of blastospore biomass by antagonistic soil-borne microorganisms. Infection potential was tested on larvae of Plutella maculipennis and Leptinotarsa decemlineata. Naked blastospores were inactivated after 3 weeks incubation, while clay-coated blastospores were still active after 2 months incubation in soil at 20°C. All investigations demonstrated the protective role of clay coating against biodegradation of fungal propagules by soil bacteria and protozoa. Consequently, clay coating of blastospores is proposed for the formulation of entomopathogenic hyphomycetes propagules. Moreover, antagonists implicated in lysis of blastospores, must be considered as an important part of the environmental response to a massive introduction of a fungus used for insect control.     

Proteins with haemagglutinin activity in larvae of the Colorado beetle, Leptinotarsa decemlineata

The haemagglutinating activity of larval haemolymph of Leptinotarsa decemlineata against red blood cells of various origins has been examined. This activity appeared to be unspecific, since all the different types of erythrocytes were agglutinated by a haemolymph dilution of $\text{1}\text{128}$ to $\text{1}\text{512}$. Only horse erythrocytes were agglutinated to a greater degree ($\text{1}\text{3200}$. Red blood cells became much more sensitive after treatment with trypsin, while formol fixation also resulted in a better agglutinability. Sulphated polysacchrides (heparin, mucin, dextran sulphate) were good inhibitors of the haemagglutination reaction. A weaker inhibition was obtained with hexosamines. As demonstrated by immunoelectrophoresis, two haemagglutinins occur in larval haemolymph. One is specific for larvae and pupae, and is therefore called the larval-pupal haemagglutinin. It is absent in adults. The second haemagglutinin is the well-known chromoprotein 2, which is present in all developmental stages, including the egg, where it constitutes an important element of yolk proteins. The affinity of chromoprotein 2 toward dextran sulphate was confirmed by precipitation tests in agarose.     

Alkane-grown Beauveria bassiana produce mycelial pellets displaying peroxisome proliferation,oxidative stress,and cell surface alterations

The entomopathogenic fungus Beauveria bassiana is able to grow on insect cuticle hydrocarbons, inducing alkane assimilation pathways and concomitantly increasing virulence against insect hosts. In this study, we describe some physiological and molecular processes implicated in growth, nutritional stress response, and cellular alterations found in alkane-grown fungi. The fungal cytology was investigated using light and transmission electron microscopy while the surface topography was examined using atomic force microscopy. Additionally, the expression pattern of several genes associated with oxidative stress, peroxisome biogenesis, and hydrophobicity were analysed by qPCR. We found a novel type of growth in alkane-cultured B. bassiana similar to mycelial pellets described in other alkane-free fungi, which were able to produce viable conidia and to be pathogenic against larvae of the beetles Tenebrio molitor and Tribolium castaneum. Mycelial pellets were formed by hyphae cumulates with high peroxidase activity, exhibiting peroxisome proliferation and an apparent surface thickening. Alkane-grown conidia appeared to be more hydrophobic and cell surfaces displayed different topography than glucose-grown cells. We also found a significant induction in several genes encoding for peroxins, catalases, superoxide dismutases, and hydrophobins. These results show that both morphological and metabolic changes are triggered in mycelial pellets derived from alkane-grown B. bassiana.     

Debilitation in conidia of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae and implication with respect to viability determinations and mycopesticide quality assessments

Germination of Beauveria bassiana (Bb) and Metarhizium anisopliae (Ma) conidia determined from a fast-rehydration (FR) protocol were compared to those obtained when dry conidia were subjected to slow rehydration (SR) by holding under high humidity conditions prior to aqueous suspension. Differences in viability estimates obtained using the FR vs. SR protocols increased markedly after conidia were exposed to various stress factors in storage (high a_w, temperature, and O₂ concentrations), with the SR protocol producing higher estimates of viability in all cases. After Bb conidia were stored under moist conditions for 21 days at 25 °C, the SR estimate of viability was >21% greater than the FR estimate. In jars flushed with different O₂ concentrations and stored at 50 °C for 34 days, proportional differences between protocols varied, depending on water activity, from 18-44% in jars flushed with 0% O₂ (100% N₂) to as high as 63-93% when treated with 21-22% O₂. For conidia stored over a broad range of moderate to high temperatures in the absence of O₂, SR-FR differences were ?9% at 25-40 °C but 30% at 50 °C. Germination of stressed Bb and Ma conidia increased substantially when incubation time on the germination substrate was increased from 24 to 72 h, whereas germination of non-stressed conidia showed little change. Conidia debilitated by stress were characterized by hypersensitivity to lethal imbibitional damage (damage that is mitigated by slow rehydration) and slow germination. Viability protocols that may provide more reliable assessments of overall mycopesticide quality are discussed.     

Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana

Entomopathogenic fungi, such as Beauveria bassiana and Metarhizium anisopliae are being developed as alternatives to chemical insecticides. They infect insects by direct penetration of the cuticle using a combination of physical pressure and extracellular hydrolytic enzymes such as proteases and chitinases. Previously we found that overexpression of a subtilisin-like protease (Pr1A) or a chitinase (Bbchit1) resulted in increased virulence of M. anisopliae and B. bassiana, respectively. In this study, we found that a mixture of the B. bassiana Pr1A homolog (CDEP1) and Bbchit1 degraded insect cuticle in vitro more efficiently than either CDEP1 or Bbchit1 alone. Based on this we produced three plasmid constructs; (1) Bbchit1, (2) CDEP1, and (3) a fusion gene of Bbchit1 linked to CDEP1 each under the control of the constitutive gpd promoter from Aspergillus nidulans. B. bassiana transformants secreting the fusion protein (CDEP1:Bbchit1) penetrated the cuticle significantly faster than the wild type or transformants overexpressing either Bbchit1 or CDEP1. Compared to the wild type, the transformant overexpressing CDEP1 showed a 12.5% reduction in LT₅₀, without a reduction in LC₅₀. The LT₅₀ of the transformant expressing CDEP1:Bbchit1 was reduced by 24.9%. Strikingly, expression of CDEP1:Bbchit1 resulted in a 60.5% reduction in LC₅₀, more than twice the reduction obtained by overexpression of Bbchit1 (28.5%). This work represents a significant step towards the development of hypervirulent insect pathogens for effective pest control.     

Gene cloning and catalysis features of a new mannitol-1-phosphate dehydrogenase (BbMPD) from Beauveria bassiana

A long-chain mannitol-1-phosphate dehydrogenase (MPD) was characterized for the first time from fungal entomopathogen Beauveria bassiana by gene cloning, heterogeneous expression and activity analysis. The cloned gene BbMPD consisted of a 1334-bp open reading frame (ORF) with a 158-bp intron and the 935-bp upstream and 780-bp downstream regions. The ORF-encoded 391-aa protein (42 kDa) showed less than 75% sequence identity to 17 fungal MPDs documented and shared two conserved domains with the fungal MPD family at the N- and C-terminus, respectively. The new enzyme was expressed well in the Luria-Bertani culture of engineered Escherichia coli BL21 by 16-h induction of 0.5 mM isopropyl 1-thio-β-d-galactopyranoside at 20 °C after 5-h growth at 37 °C. The purified BbMPD exhibited a high catalytic efficiency (k_cat/K_m) of 1.31 × 10⁴ mM⁻¹ s⁻¹ in the reduction of the highly specific substrate d-fructose-6-phosphate to d-mannitol-1-phosphate. Its activity was maximal at the reaction regime of 37 °C and pH 7.0 and was much more sensitive to Cu²⁺ and Zn²⁺ than to Li⁺ and Mn²⁺. The results indicate a crucial role of BbMPD in the mannitol biosynthesis of B. bassiana.