Attenuation of the entomopathogenic fungus Beauveria bassiana following serial in vitro transfers

Stability of pathogenicity in continuous in vitro cultivation is desirable for the purpose of large-scale production of a mycoinsecticide. Fungal biocontrol agents may lose virulence when maintain on artificial media, resulting in products of commercially inferior quality. In this research, two isolates (DEBI007 and DEBI008) of entomopathogenic fungus Beauveria bassiana were investigated for their stability following fifteen serial in vitro transfers assaying virulence to mealworm larvae, conidiation, and hyphal development on artificial culture as some fungal virulence determinants. Moreover, role of insect cuticle on fungal virulence restoration and protease 1 (Pr1) activity was considered as the most important factor. Although radial hyphal development and colony colour on in vitro culture was not affected following serial transfers, conidiation and Pr1 activity of both fungal isolates were reduced remarkably after fifteen transfers compared with control. Similarly, mean lethal concentration (LC₅₀) values were increased as the number of serial transfers on artificial diet increased, although these increases were not statistically significant in both isolates as the confidential limits of LC₅₀ values were overlapping. Our results revealed that attenuation of entomopathogenic fungi following serial in vitro transfers is a combination of interconnected factors. Other probable components such as pathogenicity determinants in this interaction should be explored in next researches.     

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.     

Interaction of Population Levels of Fusarium oxysporum f. sp. vasinfectum and Meloidogyne incognita on Cotton

In autoclaved greenhouse soil without Fusarium oxysporum f. sp. vasinfectum, Meloidogyne incognita did not cause leaf or vascular discoloration of 59-day-old cotton plants. Plants had root galls with as few as 50 Meloidogyne larvae per plant. Root galling was directly proportional to the initial nematode population level. Fusarium wilt symptoms occurred without nematodes with 77,000 fungus propagules or more per gram of soil. As few as 50 Meloidogyne larvae accompanying 650 fungus propagules caused Fusarium wilt. With few exceptions, leaf symptoms appeared sooner as numbers of either or both organisms increased. In soils infested with both organisms, the extent of fungal invasion and colonization was well correlated with the extent of nematode galling and other indications of the Fusarium wilt syndrome.     

Enzymes of the xylotrophic basidiomycete Lentinus edodes F-249 in the course of morphogenesis

It has been shown that the fungus Lentinus edodes grown on a solid wort agar substrate produces intracellular enzymes, including Mn-dependent peroxidase, laccase, and tyrosinase as a family of isoforms. The composition of the complex (containing one to four forms of each enzyme) varied during the basidiomycete life cycle. The activity of oxidases was maximal at the stage of nonpigmented mycelium and at the stages of a brown mycelial mat and a fruit body. The activity of tyrosinase increased in the course of mycelium pigmentation and had two maxima: at the stage of a brown mycelial mat and at the stage of a fruit body. Laccase and tyrosinase activities were shown to increase sharply upon addition of oak sawdust extract to the culture medium as compared with the enzyme activities of mycelium grown on wort agar alone. It was established that the effect of phenol oxidase substrates on the growing mycelium consists in a twofold acceleration of the process of morphogenesis in the fungus L. edodes.     

Acid phosphatase activity during the interaction of the nematophagous fungus Duddingtonia flagrans with the nematode Panagrellus sp

The use of Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as a biological control method against free living larvae of gastrointestinal nematodes of livestock animals. This fungus captures and infects the nematode by cuticle penetration, immobilization and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. This report characterizes the acid phosphatase activity during the interaction of D. flagrans with the free-living nematode Panagrellus sp. The optimum pH for the hydrolysis of the acid phosphatase substrate p-nitrophenyl phosphate was 2.2, 2.8 and 5.4 from D. flagrans alone and 2.2 and 5.4 for Panagrellus sp alone, fungus-nematode interaction in liquid medium and fungus-nematode interaction in solid medium. Different acid phosphatase activity bands were detected by SDS-PAGE. Maximum acid phosphatase activity of the fungus or nematode alone and of the fungus-nematode interaction occurred within 70 min of incubation in the presence of the substrate 4-methylumbelliferyl phosphate. The activity of this enzyme was significantly higher for the fungus-nematode interaction when compared to the organisms alone, indicating a synergistic response. Furthermore, structures appeared in the hyphae after 30 min, nematodes were observed adhered after 40 min and many were captured by the typical fungus traps after 70 min of interaction. The participation of acid phosphatase activity and its importance during the interaction of the fungus with the nematode were discussed.     

Optimization of growth and hydrolytic enzymes production by Fusarium culmorum using response surface method

Production of hydrolytic enzymes by a phytopathogenic fungus Fusarium culmorum was investigated. The proteolytic activity was observed when the fungus was grown in the medium containing starch or soybean meal as a carbon source. The amylolytic and lipolytic activities were not found. Response surface modeling was applied to shake-flask culture of the fungus to determine the optimum concentration of carbon source and optimal culture time for growth and protease production. The results indicated that the maximum yield of protease production corresponded to the concentration of soybean meal of 1.4?g/ml and culture time of 4.5?days. The fungus growth depends on the concentration of carbon source in the medium whereas the enzyme production was also influenced by the culture time and interaction between these two variables.     

In Vivo Characterization of Dimethylsulfoniopropionate Lyase in the Fungus Fusarium lateritium

A fungus, Fusarium lateritium, with dimethylsulfoniopropionate (DMSP) lyase activity was isolated from both seawater and a salt marsh due to its ability to grow on DMSP (with the evolution of dimethyl sulfide) as the sole source of carbon. This is the first reported case of DMSP lyase activity in a fungus. Several other common fungal genera tested did not have DMSP lyase activity. DMSP was taken up more rapidly by F. lateritium than it was utilized, leading to its intracellular accumulation. Inhibitor studies with nystatin and cyanide indicated that DMSP uptake was an energy-dependent process. The lyase was inducible by its substrate, DMSP (K_m, 1.2 mM), and by the substrate analogs choline and glycine betaine. During induction, DMSP lyase activity increased with time and then dropped rapidly. This loss of activity could be prevented by spiking the culture with fresh DMSP or choline. The V_max for DMSP lyase was 34.7 mU · mg of protein⁻¹. The inhibitory effects of nystatin, and p-chloromercuriphenylsulfonate on DMSP lyase activity suggested that the enzyme is cytosolic. Because plants like Spartina (a marsh grass) and marine algae contain high concentrations of DMSP, we speculate that DMSP-utilizing fungi may be involved in their decay.     

Zoosporicidal metabolites from an endophytic fungus Cryptosporiopsis sp. of Zanthoxylum leprieurii

Two polyketides, cryptosporiopsin A (1) and hydroxypropan-2′,3′-diol orsellinate (3), and a natural cyclic pentapeptide (4), together with two known compounds were isolated from the culture of Cryptosporiopsis sp., an endophytic fungus from leaves and branches of Zanthoxylum leprieurii (Rutaceae). The structures of these metabolites were elucidated on the basis of their spectroscopic and spectrometric data. Cryptosporiopsin A and the other metabolites exhibited motility inhibitory and lytic activities against zoospores of the grapevine downy mildew pathogen Plasmopara viticola at 10–25 μg/mL. In addition, the isolated compounds displayed potent inhibitory activity against mycelial growth of two other peronosporomycete phytopathogens, Pythium ultimum, Aphanomyces cochlioides and a basidiomycetous fungus Rhizoctonia solani. Weak cytotoxic activity on brine shrimp larvae was observed.     

Inhibition of phagocytic activity and nodulation in Galleria mellonella by the entomopathogenic fungus Nomuraea rileyi

Changes in phagocytic activity and nodulation in the greater wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae), were examined after treatment with the culture fluid of the entomopathogenic fungus Nomuraea rileyi SH1. When isolated hemocytes of G. mellonella were incubated with the conidia of N. rileyi in vitro, the rates of phagocytosis increased at 4 h after incubation but decreased subsequently. On the contrary, the rates of phagocytosis of isolated hemocytes decreased by 80% after 24 h preincubation with the fungal culture fluid (1/200, 1/100 dilutions). Levels of inhibition of phagocytic activity by the culture fluid depended on dilutions used. Galleria mellonella larvae showed a peak of nodulation at 4 h after injection with conidia. The percentage of nodules in hemolymph did not decrease by preinjection with the culture fluid, whereas the percentage of nodule‐containing conidia decreased, depending on the injected fluid. However, phagocytosis and nodulation in G. mellonella did not change after treatment of the culture fluid with proteinase K, indicating that the culture fluid contained proteinaceous immunosuppressive factors. Electrophoretic analysis of the culture fluid and not the fresh medium without culturing the fungus exhibited protein bands. Therefore, N. rileyi possibly secretes toxic proteins that impair cellular immune responses in G. mellonella larvae.     

Effect of Aspergillus flavus mycotoxin on Culex mosquito larvae

An isolate of Aspergillus flavus var. columnaris recovered from moribund larvae of the mosquito Culex peus produced metabolites which caused mortality in larvae of C. peus and C. tarsalis. Production of these toxins in vitro on solid substrate rice was based on culture techniques used in making Ang kak. The effect of incubation time on mycotoxin production was determined for cultures fermented from 3 to 13 days. Mycotoxins produced by isolate FU-051 were extracted by refluxing the spent medium with chloroform. Fractionation on a silicic acid column with a methanol-chloroform (3:97 $\text{v}\text{v}$) solvent mixture yielded two toxic fractions. Toxic activity was determined using a biological assay with mosquito larvae.     

The time--mortality response of mosquito larvae infected with the fungus Culicinomyces

In laboratory experiments with Culicinomyces, the time of death of infected Anopheles hilli larvae decreased as the concentration of conidia to which they were exposed increased. At a concentration of 10³ conidia/ml, most first instar and third instar larvae died later than 2 days after exposure and the majority of dead specimens developed external sporulation on the exterior cuticle. On the other hand, at a concentration of 10⁵ conidia/ml, more than 60% of first and third instar larvae died within 2 days of exposure. Very few of these larvae developed external sporulation and it appears that most of them died after penetration by the fungus through the gut cuticle but before the hemocoel was colonized by mycelium. The reason for this rapid death at high concentrations of inoculum is not known, but it is thought that it may be caused by toxic substances associated with the invading hyphae which only attain a lethal titer when massive invasion originates from large numbers of conidia.     

Synthesis and antifungal activity of two novel spermidine analogues

Two spermidine analogues were synthesised and examined for antifungal activity. Both compounds used as 1 mM post-inoculation sprays reduced infection of barley seedlings by the powdery mildew fungus, Erysiphe graminis f.sp. hordei, infection of broad bean seedlings by the rust fungus, Uromyces viciae-fabae, and infection of apple seedlings by the powdery mildew fungus, Podosphaera leucotricha. Since these fungal pathogens cannot be cultured axenically, the effects of the two spermidine analogues on mycelial growth in vitro, as well as preliminary investigations on polyamine biosynthesis, were undertaken using the oat stripe pathogen, Pyrenophora avenae. Although neither compound affected radial growth of the fungus on plates, both analogues reduced fungal biomass in liquid culture substantially. The two spermidine analogues, used at a concentration of 1 mM, had no significant effect on the conversion of labelled ornithine into polyamines in P. avenae.     

Screening for mycotoxins with larvae of Tenebrio molitor.

Larvae of the yellow mealworm, Tenebrio molitor, were used to screen isolates of field and storage fungi, included in their dietary substrate, for mycotoxins. Feeding of three isolates of Fusarium roseum, two of Fusarium equiseti and of Fusarium nivale, and one of an unidentified species of Fusarium resulted in growth depression of the larvae. One isolate of an unidentified species of Myrothecium was also toxic to larvae of Tenebrio molitor. Mycotoxin production was apparently dependent, not only on the fungal isolate, but also on the culture conditions under which the fungus was grown. Some fungal isolates had growth-promoting qualities for larvae of Tenebrio molitor.     

Benzylideneacetone suppresses both cellular and humoral immune responses of Spodoptera exigua and enhances fungal pathogenicity

An entomopathogenic fungus, Beauveria bassiana, had significant insecticidal activity against the beet armyworm, Spodoptera exigua. However, it took almost one week to cause significant mortality. This study used a mixture treatment with an immunosuppressant to enhance the fungal pathogenicity. A bacterial metabolite, benzylideneacetone (BZA), had a significant synergistic effect on the fungal pathogenicity against S. exigua, although it had little insecticidal activity by itself. The mixture treatment shortened median lethal time of B. bassiana by approximately 2 days. The synergistic activity of BZA on the pathogenicity of B. bassiana was induced by its immunosuppressive effects on both cellular and humoral antifungal responses of S. exigua. In response to B. bassiana, S. exigua larvae can form hemocytic nodules. Nodules were significantly suppressed by BZA treatment. Moreover, BZA inhibited expression of some antimicrobial peptide genes of S. exigua in response to fungal challenge. The immunosuppressive condition induced by BZA allowed B. bassiana to easily colonize and multiply in the hemocoel of treated larvae, which resulted in significant enhancement of the pathogenicity of B. bassiana.     

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.     

Biochemistry of mosquito infection: Preliminary studies of biochemical change in Culex pipiens quinquefasciatus following infection with Lagenidium giganteum

The effect of infection of larvae of the mosquito Culex pipiens quinquefasciatus by the fungus Lagenidium giganteum has been studied from a biochemical standpoint. Methods were developed to analyze larval extracts that were essentially free of parasitic material. Biochemical parameters investigated on a per larva basis were protein, and the enzymes o-diphenol oxidase, glutamate transaminase, alkaline phosphatase, trehalase, and chitobiase. The behavior of the total amino acid and sugar pools were also examined. In general it was found that the infected larvae exhibited a significant decrease in rate of synthesis of most of the parameters studied and that the rates eventually fell to zero when compared to healthy control organisms. The progress of mycosis was correlated with visual evidence and it was concluded that the larvae were dying from starvation as a consequence of the utilization of their endogenous reserves by the parasite. Death of the larvae occurred as a general rule, in laboratory conditions, some 48 hr after initiation of infection by the fungus.     

Biocompatibility of Steinernema glaseri with a nematode trapping fungus Arthrobotrys superba on different nutrient media

Steinernema glaseri is known to be the most efficacious biocontrol agent against many soil insects belonging to various orders and of diverse habitats. In nature the efficacy of such entomogenous nematodes may be challenged by many micro pathogens. Keeping this in view, the biocompatibility of Steinernema glaseri with a nematophagous fungus Arthrobotrys superba was assessed on eight different nutrient media. The predatory activity of A. superba was evaluated in terms of trap formation, conidiophore formation, and number of adhesive cells formed in the presence and absence of nematodes. The fungus failed to form any trap on any of the culture media in the absence of nematodes. However, in the presence of nematodes, the trap formation by the A. superba was increased and found to be maximum on LNM (low nutrient mineral salt) medium and minimum at WA (water agar) of all the culture media tested. In the present investigation, the effect of CMA (2% and 5%) was not considerable; however, gradually higher dilutions of CMA induced greater number of traps. This less pronounced effect might be due to nutrient richness because low nutrient levels were found to be necessary for the formation of conidial traps. The number of conidiophores decreased with increase in dilution of Corn Meal Agar from 5% to 2%. Higher number of chlamydospores was observed in phenylalanine treated medium which indicates the inhibiting effect of phenylalanine on the growth of A. superba. Our results suggest that richness of soil has pronounced effect on the predatory activity of nematophagous fungus, Arthrobotrys superba which may further affect the biopotential of entomogenous nematode Steinernema glaseri, therefore, care should be taken while releasing entomogenous nematodes in an agroecosystem for managing various insect pests in a more efficient manner.     

A mosquito parasite from a mosquito predator

The fungus Coelomomyces macleayae has been reported from treehole mosquito larvae of three Aedes subgenera in Australia, Fiji, and the United States. This fungus is now recorded for the first time from a mosquito of the genus Toxorhynchites, the large predatory larvae of which are of some importance in the naturalistic control of associated mosquito pests and vectors. A single parasitized larva of T. rutilus septentrionalis was collected from a magnolia treehole (previously used for A. triseriatus infection experiments) near West Lake, Louisiana, in September, 1971. Larval A. triseriatus and Orthopodomyia signifera were also present but were uninfected.     

Metarhizium anisopliae Pathogenesis of Mosquito Larvae: A Verdict of Accidental Death

Metarhizium anisopliae, a fungal pathogen of terrestrial arthropods, kills the aquatic larvae of Aedes aegypti, the vector of dengue and yellow fever. The fungus kills without adhering to the host cuticle. Ingested conidia also fail to germinate and are expelled in fecal pellets. This study investigates the mechanism by which this fungus adapted to terrestrial hosts kills aquatic mosquito larvae. Genes associated with the M. anisopliae early pathogenic response (proteinases Pr1 and Pr2, and adhesins, Mad1 and Mad2) are upregulated in the presence of larvae, but the established infection process observed in terrestrial hosts does not progress and insecticidal destruxins were not detected. Protease inhibitors reduce larval mortality indicating the importance of proteases in the host interaction. The Ae. aegypti immune response to M. anisopliae appears limited, whilst the oxidative stress response gene encoding for thiol peroxidase is upregulated. Cecropin and Hsp70 genes are downregulated as larval death occurs, and insect mortality appears to be linked to autolysis through caspase activity regulated by Hsp70 and inhibited, in infected larvae, by protease inhibitors. Evidence is presented that a traditional host-pathogen response does not occur as the species have not evolved to interact. M. anisopliae retains pre-formed pathogenic determinants which mediate host mortality, but unlike true aquatic fungal pathogens, does not recognise and colonise the larval host.