Variations in UV-B tolerance and germination speed of Metarhizium anisopliae conidia produced on insects and artificial substrates

Solar ultraviolet radiation (UV-A and UV-B) is a major factor in failure of programs using the insect pathogenic fungus Metarhizium anisopliae as a biological control agent. Studies were conducted to determine if growth conditions, viz. artificial (agar media or rice grain) or natural (infected insects) substrates for conidial production affect two traits that directly influence performance of conidia after field application: tolerance to UV-B radiation and conidial germination speed. Conidia of two isolates (ARSEF 23 and ARSEF 2575) of M. anisopliae var. anisopliae produced on potato dextrose agar plus yeast extract (PDAY) or on fungus-killed larvae of two insect species, Galleria mellonella and Zophobas morio, were inactivated by exposure to UV-B radiation. Conidia of both isolates when produced on insect cadavers were significantly more sensitive to UV-B radiation than conidia produced on PDAY. Also, conidia from insect cadavers germinated slower than those from PDAY cultures. A comparison of conidia from artificial substrates showed that conidia produced on Czapek's and Emerson's YpSs agar media or rice grains had higher tolerance to UV-B radiation and germinated faster than conidia raised on PDA and PDAY. Accordingly, the growth substrate and nutritional environment in which conidia are produced influences M. anisopliae conidial UV-B tolerance and speed of germination; and manipulation of these variables could be used to obtain conidia with increased tolerance to UV-B radiation and shorter germination times.     

Different defense strategies of Dendrolimus pini, Galleria mellonella, and Calliphora vicina against fungal infection

The resistance of Galleria mellonella, Dendrolimus pini, and Calliphora vicina larvae against infection by the enthomopathogen Conidiobolus coronatus was shown to vary among the studied species. Exposure of both G. mellonella and D. pini larvae to the fungus resulted in rapid insect death, while all the C. vicina larvae remained unharmed. Microscopic studies revealed diverse responses of the three species to the fungal pathogen: (1) the body cavities of D. pini larvae were completely overgrown by fungal hyphae, with no signs of hemocyte response, (2) infected G. mellonella larvae formed melanotic capsules surrounding the fungal pathogen, and (3) the conidia of C. coronatus did not germinate on the cuticle of C. vicina larvae. The in vitro study on the degradation of the insect cuticle by proteases secreted by C. coronatus revealed that the G. mellonella cuticle degraded at the highest rate. The antiproteolytic capacities of insect hemolymph against fungal proteases correlated well with the insects' susceptibility to fungal infection. The antiproteolytic capacities of insect hemolymph against fungal proteases correlated well with the insects' susceptibility to fungal infection. Of all the tested species, only plasmatocytes exhibited phagocytic potential. Exposure to the fungal pathogen resulted in elevated phagocytic activity, found to be the highest in the infected G. mellonella. The incubation of insect hemolymph with fungal conidia and hyphae revealed diverse reactions of hemocytes of the studied insect species. The encapsulation potential of D. pini hemocytes was low. Hemocytes of G. mellonella showed a high ability to attach and encapsulate fungal structures. Incubation of C. vicina hemolymph with C. coronatus did not result in any hemocytic response. Phenoloxidase (PO) activity was found to be highest in D. pini hemolymph, moderate in G. mellonella, and lowest in the hemolymph of C. vicina. Fungal infection resulted in a significant decrease of PO activity in G. mellonela larvae, while that in the larvae of D. pini remained unchanged. PO activity in C. vicina exposed to fungus slightly increased. The lysozyme-like activity increased in the plasma of all three insect species after contact with the fungal pathogen. Anti E. coli activity was detected neither in control nor in infected D. pini larvae. No detectable anti E. coli activity was found in the control larvae of G. mellonella; however, its exposure to C. coronatus resulted in an increase in the activity to detectable level. In the case of C. vicina exposure to the fungus, the anti E. coli activity was significantly higher than in control larvae. The defense mechanisms of D. pini (species of economic importance in Europe) are presented for the first time.     

To complete their life cycle, pathogenic nematode-bacteria complexes deter scavengers from feeding on their host cadaver

The life cycle of commercially used molluscicidal rhabditid nematodes Phasmarhabditis hermaphrodita and entomopathogenic steinernematid nematodes is similar: infective stages carry symbiotic bacteria, which kill their host. Nematodes complete their life cycle feeding on the proliferating symbiont and the host tissue. After 1-2 weeks, new infective stages carrying the bacteria leave the host cadaver in search of new hosts. The removal of invertebrate cadavers by scavengers is extremely fast and represents a severe threat to the developing nematodes.Two-choice trials were used to assess prey choice of the generalist predator/scavenger Pterostichus melanarius (Coleoptera: Carabidae) between Deroceras reticulatum (Mollusca: Agriolimacidae) slugs or wax moth Galleria mellonella (Lepidoptera: Pyralidae) larvae killed by infection of P. hermaphrodita/Steinernema affine and control killed by freezing. We demonstrate that the presence of either of the two nematodes tested deters the beetles from consuming infected cadavers. As P. hermaprodita cannot infect an insect host, we hypothesise the deterrent effect being an evolutionary adaptation of the nematode/bacteria complex rather than the ability of the beetles to avoid potentially infective cadavers.     

Fusarium pathogenesis investigated using Galleria mellonella as a heterologous host

Members of the fungal genus Fusarium are capable of manifesting in a multitude of clinical infections, most commonly in immunocompromised patients. In order to better understand the interaction between the fungus and host, we have developed the larvae of the greater wax moth, Galleria mellonella, as a heterologous host for fusaria. When conidia are injected into the haemocoel of this Lepidopteran system, both clinical and environmental isolates of the fungus are able to kill the larvae at 37 °C, although killing occurs more rapidly when incubated at 30 °C. This killing was dependent on several other factors besides temperature, including the Fusarium strain, the number of conidia injected, and the conidia morphology, where macroconidia are more virulent than their microconidia counterpart. There was a correlation in the killing rate of Fusarium spp. when evaluated in G. mellonella and a murine model. In vivo studies indicated G. mellonella haemocytes were capable of initially phagocytosing both conidial morphologies. The G. mellonella system was also used to evaluate antifungal agents, and amphotericin B was able to confer a significant increase in survival to Fusarium-infected larvae. The G. mellonella-Fusarium pathogenicity system revealed that virulence of Fusarium spp. is similar, regardless of the origin of the isolate, and that mammalian endothermy is a major deterrent for Fusarium infection and therefore provides a suitable alternative to mammalian models to investigate the interaction between the host and this increasingly important fungal pathogen.     

Effects of environmental factors on virulence of the entomopathogenic fungus, Nomuraea rileyi, against the corn earworm, Helicoverpa armigera (Lep., Noctuidae)

The effects of environmental factors on infection of the entomopathogenic fungus, Nomuraea rileyi , isolated from the corn earworm, Helicoverpa armigera , in Taiwan, to its host insect were studied in the laboratory. The fungus caused higher larval mortality at 20°C than at 30°C when 5 × 10⁶ conidia/ml were sprayed on the fourth instar. However, mortality of the fifth instar injected with 1 × 10³ conidia/larva was not significantly different when the inoculated larvae were incubated from 15 to 30°C. The fungal development in inoculated larvae was best at 20 and 25°C after shifting from 20°C to either lower or higher temperatures. The germination rate was higher at 20 and 25°C than at 30 or 35°C. Conidial germination was better on the wash-off of insect cuticle than on Sabouraud maltose agar with yeast extract. Sporulation on chill-dried cadavers was maximal at 95 or 100% relative humidity than at lower levels of relative humidity. The time required for sporulation was 2 days less at 100% than at 95% relative humidity. Although photoperiod did not affect fifth instar mortality caused by N. rileyi , the median lethal time (LT₅₀) values were shorter upon incubating under light than in darkness. Incubation of infected cadavers under 12 or 24 h light resulted in 20-fold more conidial production than under full darkness. Therefore, illumination is necessary for development of this isolate on insect cadavers.     

Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae

Candida albicans is a dimorphic human pathogen in which the yeast to hyphal switch may be an important factor in virulence in mammals. This pathogen has recently been shown to also kill insects such as the Greater Wax Moth Galleria mellonella when injected into the haemocoel of the insect larvae. We have investigated the effect of previously characterised C. albicans mutations that influence the yeast to hyphal transition on virulence in G. mellonella larvae. There is a good correlation between the virulence of these mutants in the insect host and the virulence measured through systemic infection of mice. Although the predominant cellular species detected in G. mellonella infections is the yeast form of C. albicans, mutations that influence the hyphal transition also reduce pathogenicity in the insect. The correlation with virulence measured in the mouse infection system suggests that Galleria may provide a convenient and inexpensive model for the in vivo screening of mutants of C. albicans.     

感染松墨天牛的金龟子绿僵菌菌株的初步筛选

松墨天牛是松材线虫病的主要媒介昆虫,本文对分离自松墨天牛的6株金龟子绿僵菌,以及从光肩星天牛、大蜡螟、伊藤厚丝角叶蜂和土蝽上分离的各1个菌株,共10个菌株的产孢情况、孢子萌发率进行研究;在此基础上选出金龟子绿僵菌1291、1349和2049三个菌株及球孢白僵菌F-263菌株,采用成虫跗节接种法和幼虫浸渍法进行室内松墨天牛及大蜡螟的毒力测定比较。结果表明,供试绿僵菌菌株对松墨天牛幼虫在接种15天后的感染僵虫率为76.9％～93.1％(1×10⁷孢子/mL);成虫在接种20天后的僵虫率亦达57.9％～75.0％(6.5×10⁵～3.4×10⁶孢子/成虫);2049菌株表现尤其突出;对应的球孢白僵菌F-263对幼虫和成虫的僵虫率分别是96.3％(1×10⁷孢子/mL)和55％(9.7×10⁵孢子/成虫)。但这3个金龟子绿僵菌菌株对大蜡螟的毒力较低,存在较明显的寄主专化性。这3个菌株今后在防治松墨天牛方面具有较大开发应用价值。     

Facultative scavenging as a survival strategy of entomopathogenic nematodes

Entomopathogenic nematodes cannot be considered only as parasitic organisms. With dead Galleria mellonella larvae, we demonstrated that these nematodes use scavenging as an alternative survival strategy. We consider scavenging as the ability of entomopathogenic nematodes to penetrate, develop and produce offspring in insects which have been killed by causes other than the nematode-bacteria complex. Six Steinernema and two Heterorhabditis species scavenged but there were differences among them in terms of frequency of colonisation and in the time after death of G. mellonella larvae that cadavers were penetrated. The extremes of this behaviour were represented by Steinernema glaseri which was able to colonise cadavers which had been freeze-killed 240 h earlier and Heterorhabditis indica which only colonised cadavers which had been killed up to 72 h earlier. Also, using an olfactometer, we demonstrated that entomopathogenic nematodes were attracted to G. mellonella cadavers.     

Influence of temperature preferences of two Paecilomyces fumosoroseus lineages on their co-infection pattern

In order to clarify the epidemiological potential of entomopathogenic fungi for insect pest control, the role of the temperature as one environmental constraint was investigated on the pattern of co-infection of Galleria mellonella by two distinct lineages of a hyphomycete, Paecilomyces fumosoroseus. The distribution of conidial populations collected on cadavers of hosts co-infected under 20 regimes, ranging from 13 to 35 degrees C, was examined. The apparent temperature tolerance of both fungal isolates was related to their in vitro colony growth and their in vivo sporulation ability. The conidial populations were characterized by molecular markers based on restriction fragment length polymorphisms of the internal transcribed spacers (ITS-RFLP) and random amplified polymorphic DNA (RAPD) contrasting profiles in combination with the conidial size. This study allowed a different temperature profile was identified for each isolate. Under most temperature regimes, only one lineage prevailed upon the infected insect; whereas both lineages coexisted at 20-25 and 25-25 degrees C. When one haplotype dominated, the displacement of the other one depended on its temperature tolerance. These results suggest that more consideration should be given to population-genetics analyses for evaluating the adaptability of microbial control agents to targeted environments.     

Venturia canescens parasitizing Galleria mellonella and Anagasta kuehniella: differing suitability of two hosts with highly variable growth potential

Venturia canescens (Grav.) (Hymenoptera: Ichneumonidae) is a solitary larval koinobiont endoparasitoid, ovipositing in several larval instars of different pyralid moth species that are pests of stored food products. After oviposition, the host larva continues to feed and grow for at least several days, the precise time doing so depending on the stage attacked. We examined the relationship between host stage and body mass on parasitoid development in late second to fifth instars of two hosts with highly variable growth potential: the wax moth, Galleria mellonella (L) and the flour moth, Anagasta kuehniella (Zeller)(Lepidoptera: Pyralidae). G. mellonella is the largest known host of V. canescens, with healthy larvae occasionally exceeding 400mg at pupation, whereas those of A. kuehniella rarely exceed 40 mg at the same stage. Parasitoid survival was generally higher in early instars of G. mellonella than in later instars. By contrast, percentage adult emergence in A. kuehniella was highest in late fifth instar and lowest in late second instar. A. kuehniella was the more suitable host species, with over 45% adult emergence in all instars, whereas in G. mellonella we found less than 35% adult emergence in all instars. Adult parasitoid size increased and egg-to-adult development time decreased in a host size- and instar-specific manner from A. kuehniella. The relationship between host size and stage and these fitness correlates was less clear in G. mellonella. Although both host species were parasitized over a similar range of fresh weights, the suitability weight-range of A. kuehniella was considerably wider than G. mellonella for the successful development of V. canescens. However, in hosts of similar weight under 5 mg when parasitized, larger wasps emerged from G. mellonella than from A. kuehniella. Parasitoid growth and development is clearly affected by host species, and we argue that patterns of host utilization and resource acquisition by parasitoids have evolved in accordance with host growth potential and the nutritional requirements of the parasitoid.     

In vitro rearing of the parasitoid Exorista larvarum (Diptera: Tachinidae) from eggs laid out of host

We evaluated the possibility of using the eggs laid out of host to rear Exorista larvarum (L.) (Diptera: Tachinidae), a larval parasitoid of Lepidoptera, on artificial media. In a first experiment, eggs oviposited on a plastic sheet (either by inexperienced or experienced females) showed the same in vitro hatching capability as those removed from the larvae of the factitious host Galleria mellonella L. Subsequently, eggs laid on the host integument or out of host, either by inexperienced or experienced females, were removed from the oviposition substrate and placed on a skimmed milkbased artificial medium. The percentages of hatched eggs, of puparia and adults, as well as the puparial weights did not differ significantly among the three treatments. These findings suggested that E. larvarum may be successfully reared in vitro with a total exclusion of the host insect. In a further test, no difference for the in vitro hatching time was found between the eggs oviposited either on a plastic sheet or on G. mellonella larvae in the same length of time (60 min). This result suggested that at oviposition the out-of-host eggs were unincubated, similarly to those that had been laid on the host larvae.     

Mortality of Delia floralis, Galleria mellonella and Mamestra brassicae treated with insect pathogenic hyphomycetous fungi

Abstract:  The susceptibility of Delia floralis eggs, neonates and larvae and the susceptibility of Galleria mellonella and Mamestra brassicae larvae to seven different Norwegian isolates of the insect pathogenic, hyphomycetous fungi Tolypocladium cylindrosporum , Metarhizium anisopliae and Beauveria bassiana , were investigated. Metarhizium anisopliae isolate ARSEF 5520 was highly virulent to G. mellonella larvae and caused 100% mortality when tested at a concentration of 3.6 × 10⁶ conidia/ml. The same M. anisopliae isolate was not virulent to D. floralis larvae. Isolates of T.cylindrosporum , were equally virulent to G. mellonella and D. floralis causing up to 36.0% mortality of larvae. It is suspected, however, that the use of grated rutabaga as a food source in the D. floralis bioassay reduced the fungal virulence of both M. anisopliae and T. cylindrosporum to D. floralis . Among three T. cylindrosporum isolates tested at a concentration of 1.0 × 10⁷ conidia/ml against eggs of D. floralis none of them reduced the hatching percentage. One isolate, ARSEF 5525 did, however, significantly reduce the longevity of neonates. Beauveria bassiana isolates ARSEF 5510 and ARSEF 5370 tested at a concentration of 1.0 × 10⁷ conidia/ml resulted in M. brassicae mortality levels of 70.0 and 55.0%, respectively. The B. bassiana isolate ARSEF 5557, however, was not virulent to M. brassicae . Among the three isolates tested against M. brassicae the two virulent isolates produced a red pigment, probably oosporein, when cultured in Sabouraud dextrose agar.     

Exoproteinases of the type A in pathogenesis of insect bacterial diseases

Immune inhibitors produced in infected larvae of Galleria mellonella by such entomopathogens as Pseudomonas aeruginosa, Serratia marcescens and Heterorhabditis bacteriophora effectively blocked in vitro bactericidal activity of insect haemolymph against Escherichia coli D31, both in Galleria mellonella and Pieris brassicae pupae previously vaccinated with Enterobacter cloacae. Even at a trace concentration, the extracellular proteinases, by proteolytic degradation, totally destroyed the activity of cecropin peptides from Galleria and cecropin-like and attacin-family proteins from Pieris, but no ability to destroy antibacterial activity was shown by extracts obtained from Galleria larvae killed by massive doses of bacterial saprophytes. It is suggested that by blocking antibacterial immune response of the host, the proteinases help the bacteria to multiply in the haemolymph, thus they could be considered an important factor in the pathogenesis of bacterial diseases of insects.     

Interaction of microbial populations in Steinernema (Steinernematidae,Nematoda) infected Galleria mellonella larvae

Infection of Galleria mellonella larvae with the entomopathogenic nematodes Steinernema feltiae (A21 and R strains) and Steinernema glaseri (Dongrae) resulted in several species of bacteria, including the respective bacterial symbiont, Xenorhabdus spp., growing in the infected insect cadavers. These other bacteria were Enterococcus in all three nematode infections studied and Acinetobacter in the S. feltiae infections. The respective populations of these bacteria changed with time. Following infection of G. mellonella larvae with any one of the Steinernema sp., only Enterococcus bacteria were detected initially in the dead larvae. Between 30 and 50h post-infection Xenorhabdus bacteria were detected and concurrent with this Enterococcus population declined to zero. This was probably due to secondary metabolites with antibacterial properties that were produced by Xenorhabdus. In the S. feltiae (both R and A21 strains) infections a third bacterium, Acinetobacter, appeared at about 130h (in S. feltiae A21 infections) or 100h (in S. feltiae R infections) and increased in population size to approximately that of Xenorhabdus. It was demonstrated that Enterococcus, orginating from the G. mellonella digestive tract, was sensitive to the organically soluble antimicrobials produced by Xenorhabdus but Acinetobacter, which was carried by the nematode, was not.     

拟双角斯氏线虫D43品系鞘蛋白对大蜡螟幼虫的免疫抑制作用

侵染期的拟双角斯氏线虫Steinernema ceratophorum D43品系体外都包裹着一个透明的体鞘。为探明体鞘对线虫侵染力的影响, 了解鞘蛋白（sheath proteins, SPs）对大蜡螟Galleria mellonella 幼虫的免疫抑制作用, 本研究通过化学方法使拟双角斯氏线虫D43脱鞘, 以对寄主的致死率和侵入点数量为指标, 与包鞘线虫比较对大蜡螟幼虫的侵染力; 采用乙醇提取的方法获得线虫鞘蛋白, 利用双向电泳和质谱技术对鞘蛋白进行鉴定分析; 从血细胞数量和酚氧化酶活力两个方面评价鞘蛋白对大蜡螟幼虫免疫反应的抑制作用。结果表明： 0.5%次氯酸钠处理20 min可以保证95%以上的线虫存活和脱鞘。与包鞘线虫相比, 脱鞘线虫对大蜡螟幼虫的致死率显著降低, 致死时间延后, 节间膜侵入点数量显著减少。以35%乙醇提取的鞘蛋白提取物可鉴定出6种鞘蛋白, 其中一个被鉴定为丝氨酸蛋白酶。此外, 血腔注射鞘蛋白提取物可导致试虫血细胞数量明显降低, 酚氧化酶活力受到显著抑制。由此说明, 体鞘对拟双角斯氏线虫D43的侵染力具有显著影响, 鞘蛋白在抑制寄主昆虫免疫反应中发挥重要作用。     

Characterization of Spanish strains of Verticillium lecanii

We have characterized biologically and physiologically eight Verticillium lecanii strains from several origins including insect pests. Of all the temperatures tested, 25 degrees C was the best for growth and at 40 degrees C none of the strains could grow. At 4 and 7 degrees C, growth was reduced in comparison to warmer temperatures. The strains had better development at pH close to 7 (F = 27.64, P < 0,01) than at pH 3. Self-inhibition of germination of strain 50 was found when more than 0.78 conidia/cm(2) were plated on corn meal agar (CMA). Germination of conidia was close to 100% for all strains except one, three days after inoculation. Among extracellular enzymatic activities studied the fungal strains showed strongest proteolytic activities followed by lipolytic and chitinolytic activities. Some strains showed significant differences (P < 0.05) in conidia production. Most of the fungicides tested (especially benomyl) inhibited radial growth of strain 50 on CMA. Pathogenicity (as median lethal time, LT50) of V. lecanii strains on larvae of Galleria mellonella varied from 2.66 -/+ 0.33 to 4.27 -/+ 0.25 days. We conclude that in vitro tests per se are not sufficient to select the best biocontrol strains of entomopathogenic fungi. Pathogenicity is a complex process in which the presence, timing and regulation of many factors including those covered in this paper, as well as their interactions, are probably involved.     

Inhibition of Beauveria bassiana Proteases and Fungal Development by Inducible Protease Inhibitors in the Haemolymph of Galleria mellonella Larvae

Injection of zymosan or dead yeast cells enhanced the inhibitory activity against exocellular Beauveria bassiana proteases in the cell - free haemolymph of Galleria mellonella larvae . Pre - injected larvae exhibited no decreased mortality after subsequent injection with living B. bassiana blastospores but survived for a prolonged time before death . Increased levels of protease inhibitors in the haemolymph were also observed after injection of B. bassiana proteases . In contrast , no enhanced inhibitory activity against B. bassiana proteases was detected in infected larvae when mycosis was initiated with conidia which enabled the fungus to invade host larvae through the integument in a natural manner . B. bassiana proteases were not completely inhibited by the addition of cell - free haemolymph . Protease inhibitors obtained after heat and trichloroacetic acid precipitation of cell - free haemolymph were added to the protein medium of B. bassiana to study the effect on its growth in vitro. Enriched fractions from pre - injected larvae delayed fungal growth in comparison with fractions from untreated larvae , suggesting that delayed mortality of immunized G. mellonella larvae infected with B. bassiana is due to enhanced levels of protease inhibitors . A non - virulent form of the same strain exhibited reduced capacity to release proteases in vitro. The results strongly suggest that the capacity of insects to release inhibitors against fungal proteases influences their susceptibility against entomopathogenic fungi .     

Generation and properties of a luminescent insect pathogen Xenorhabdus nematophilus (Enterobacteriaceae)

Studies on the interaction of the insect pathogenic bacterium, Xenorhabdus nematophilus (Enterobacteriaceae), with its nematode and insect hosts would be greatly assisted if a luminescent phenotype were generated that would allow the detection of viable bacteria in vivo without the necessity for disruption of the cellular interactions. The plasmid, pMGM221, containing the luminescence gene (luxCDABE) of Vibrio harveyi was introduced into different strains (DD136 and 19061) and phases (one and two) of X. nematophilus by triparental mating. For reproducible and efficient conjugation, it was necessary to use older cultures (96-160 h) in the stationary phase of X. nematophilus for mating with relatively small differences (<2-fold) in transconjugant yield for the different strains and phases of X. nematophilus. All transconjugants emitted high levels of light with optimum bioluminescence at 27 degrees C in Luria broth at pH 8.0 containing 20 g/L NaCl; pH, osmolarity, and temperature conditions were similar to those encountered by the bacteria in the hemolymph of the larvae of Galleria mellonella. Plasmids were detected in the transconjugants after 6 months of subculturing the bacteria without antibiotic selection. Aside from light emission, luminescent transconjugants had the same physiological properties as the nonluminescent parental strains, including identical rates of growth, production of exoenzymes, removal from and subsequent emergence into the insect's hemolymph, bacterial-induced hemocyte damage, suppression of prophenoloxidase activation, and the ability to kill G. mellonella larvae. Light-emitting larvae could readily be detected by eye in a dark room, and all bacteria reisolated from dead larvae were luminescent. These properties validate the use of luminescent X. nematophilus not only as a means of following bacterial host interactions, but also as a potential agent to follow the infection and death of the insect population.     

Lack of host specialization in Aspergillus flavus

Aspergillus spp. cause disease in a broad range of organisms, but it is unknown if strains are specialized for particular hosts. We evaluated isolates of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans for their ability to infect bean leaves, corn kernels, and insects (Galleria mellonella). Strains of A. flavus did not affect nonwounded bean leaves, corn kernels, or insects at 22 degrees C, but they killed insects following hemocoelic challenge and caused symptoms ranging from moderate to severe in corn kernels and bean leaves injured during inoculation. The pectinase P2c, implicated in aggressive colonization of cotton balls, is produced by most A. flavus isolates, but its absence did not prevent colonization of bean leaves. Proteases have been implicated in colonization of animal hosts. All A. flavus strains produced very similar patterns of protease isozymes when cultured on horse lung polymers. Quantitative differences in protease levels did not correlate with the ability to colonize insects. In contrast to A. flavus, strains of A. nidulans and A. fumigatus could not invade living insect or plant tissues or resist digestion by insect hemocytes. Our results indicate that A. flavus has parasitic attributes that are lacking in A. fumigatus and A. nidulans but that individual strains of A. flavus are not specialized to particular hosts.     

Expression of pathogenesis-related genes in Metarhizium anisopliae when infecting Spodoptera exigua

Characterization of pathogenesis genes of Metarhizium anisopliae, will provide better understanding of the role of these genes during pathogenesis. The expression profiles of pathogenesis-related genes encoding for a subtilisin-like protease (PR1), two types of chitinases (CHI2 and CHI3), and a peptide synthetase (PES) were studied during the different stages of M. anisopliae infection in Spodoptera exigua larvae using quantitative real-time RT-PCR. Sampling were at 0, 2, 12, and 24 h after infection, when the infected larvae reached the moribund stage (36 h), when mycelia emerged from the cadavers, when few conidia had formed on the mycelia, and when the cadavers were covered by conidia. For comparison, conidia and mycelial samples harvested from culture media were also included. Among the studied genes, PR1 expression was detected early at 2 h after infection and increased as the infection progressed. CHI2 and CHI3 expressions were detected 12 h after infection and when the mycelia emerged from cadavers, respectively. The expression levels of PR1, CHI2 and CHI3 genes increased significantly at the beginning of conidiogenesis on cadavers, but decreased at later stages. As expected, their expressions in pure fungal propagules were at very low levels. For PES gene, fold changes were not significant between different samples (less than onefold), indicating it might not have a major role in infecting stages. High expression levels of PR1, CHI2, and CHI3 genes during the post-mortem hyphal growth and conidiation stages of M. anisopliae clearly indicate the importance of these genes during the saprophytic phase of this fungus on host insect.