Fungal Conidia Research Articles

Effect of storage conditions (relative humidity, duration, and temperature) on the germination time of Aspergillus carbonarius and Penicillium chrysogenum

Fungal conidia are disseminated, often in the air, for a certain period of time, prior to contaminating food products. The objective of this study was to examine the effects of the relative humidity, RH (%), time (day), and temperature (°C) during this period of time, called “storage”, on the germination time, τ (h), of Aspergillus carbonarius and Penicillium chrysogenum. A Doehlert design was used in the range, 20–100% RH, 2–28days, and 5–25°C. As compared to un-stored conidia, the germination time of conidia stored at 60% RH, 15days, 5°C was increased by 23 and 28%, for A. carbonarius and P. chrysogenum, respectively. Stored conidia exhibited a minimum τ value at 60% RH, and 100% RH for A. carbonarius and P. chrysogenum, respectively. For these species, τ was minimum for 2days of storage. The effect of temperature was RH dependent for A. carbonarius. The germination time of stored conidia was clearly greater than that of fresh conidia obtained in the laboratory. This result should be taken into account to determine the mould free shelf-life of food products.

P144 Pathogen recognition by the long pentraxin PTX3: Roles of glycosylation and interplay with complement

Introduction The long pentraxin PTX3 is a soluble Pattern Recognition Molecule (sPRM) with non-redundant roles in resistance to selected pathogens, such as the opportunistic fungus Aspergillus fumigatus . PTX3 facilitates recognition and phagocytosis of fungal conidia mainly by polymorphonuclear neutrophils (PMNs) via Fc γ receptors (Fc γ Rs) and alternative complement pathway (ACP) mechanisms. The human PTX3 protein contains a single N-glycosylation site with complex type sialylated oligosaccharides that has been involved in a number of PTX3 functions [1] . The present study was designed to characterize the role of this post translational modification in the PTX3-depedent recognition and phagocytosis of A. fumigatus . This microorganism has developed several strategies to evade the immune response, such as attracting onto the conidial wall inhibitors of the ACP, like factor H (FH, a ligand of PTX3). Here we present preliminary evidences on the effect of PTX3 on FH engagement by A. fumigatus . Methods Recombinant as well as natural human PTX3 (i.e., from PMNs) was purified by immunoaffinity chromatography and the attached oligosaccharides were analyzed using glycosidases, lectin blotting and mass spectrometry. A range of PTX3 glycoforms were made in vitro by enzymatic hydrolysis of terminal monosaccharides of the recombinant protein. The opsonic and pro-phagocytic activities of these variants were characterized in buffer solutions and whole human peripheral blood, respectively, by flow cytometry. The influence of PTX3 on FH binding to conidia was studied in competition assays using microtiter plates. Results Complex-type sugars were found on PTX3 of both natural and recombinant origins. These showed heterogeneity with regard to the relative content of terminal sialic acid (SA) and bi-, tri- and tetrantennary glycans, depending on the cellular source. Enzymatic removal of SA caused ∼50% reduction of PTX3 binding to A. fumigatus conidia, with no further change due to additional deglycosylation. Conversely, saccharide hydrolysis had no effect on PTX3 binding to PMNs, where Fc γ Rs have been proposed as putative receptors of this pentraxin [1] . However, consistent with binding data from conidia, desialylated PTX3 showed reduced potentiation of conidia phagocytosis by PMNs as compared to the unmodified protein. Also, we observed that PTX3 inhibits the binding of FH to A. fumigatus conidia, even when these were pre-opsonised with the long pentraxin and FH was applied at physiologically relevant concentrations. Conclusion Different pathogens express neuraminidases on their surface during infection and natural immunity cells, such as PMNs, mobilize intracellular neuraminidases to the plasma membrane upon inflammation. Here, we report that the glycosylation status of the sPRM PTX3 changes, with respect to SA, depending on cellular source and perhaps inducing stimuli. We describe that SA modulates the PTX3-dependent recognition and phagocytosis of A. fumigatus , which points to glycosylation as a strategy to tune PTX3 functions in innate immunity. Furthermore, we provided evidences that PTX3 impairs the interaction of FH with A. fumigatus , where this might link to a new mechanism to counterbalance the complement evasion strategy exploited by this pathogen.

Genetic variation of Fusarium oxysporum f. sp. lycopersici isolated from tomatoes in Thailand using pathogenicity and AFLP markers

Genetic variation among 25 isolates of Fusarium oxysporum f. sp. lycopersici causing tomato wilt was determined using pathogenicity test and Amplified Fragment Length Polymorphisms (AFLP) markers. The isolates were collected from 8 provinces in Thailand. Based on the pathogenecity result, all isolates were divided into two groups as pathogenic and non-pathogenic isolates. Cluster analysis based on AFLP also grouped the pathogenic isolates into 3 subgroups as low, moderate and high virulence. A dendrogram resulting from a cluster analysis showed two main distinct groups: group 1, non-pathogenic isolates; and group 2, pathogenic isolates rooting from outgroup. Eighty one polymorphic bands were analyzed using computer software. The results showed that genetic differentiation occurred among populations (Gst = 0.5898). However, the populations in the same geographical areas, Khonkaen and Nong khai, Tak and Phechaboon were more closely related genetically than another populations based on Nei’s genetic distance, indicating the movement of the fungal conidia between these areas. This work provided new information on the formae speciales of F. oxysproum f splycopersici NKSC01 and NKSC02, of high virulent which could be classified as race 2 causing wilt of tomato var. Cheery. Key words: Fragment length polymorphisms (AFLP) marker, tomato, pathogenecity,Fusarium oxysporum f. sp. Lycopersici.

Recognition of a core fragment ofBeauveria bassiana hydrophobin gene promoter (P hyd1) and its special use in improving fungal biocontrol potential.

To identify a suitable promoter for use in engineering fungal entomopathogens to improve heterologous gene expression and fungal biocontrol potential, a 1798 bp promoter (Phyd1) upstream of Beauveria bassiana class I hydrophobin gene (hyd1) was optimized by upstream truncation and site-directed mutation. A truncated 1290 bp fragment (Phyd1-t1) drove eGFP expression in B. bassiana much more efficiently than full-length Phyd1. Further truncating Phyd1-t1 to 1179, 991 and 791 bp or mutating one of the binding domains of three transcription factors in Phyd1-t1 reduced significantly the expression of eGFP (enhanced green fluorescence protein). Under Phyd1-t1 control, eGFP was expressed more abundantly in conidiogenic cells and conidia than in mycelia. Therefore, Phyd1-t1 was used to integrate a bacterium-derived, insect midgut-specific toxin (vip3Aa1) gene into B. bassiana, yielding a transgenic strain (BbHV8) expressing 9.8-fold more toxin molecules in conidia than a counterpart strain (BbV28) expressing the toxin under the control of PgpdA, a promoter widely used for gene expression in fungi. Consequently, BbHV8 showed much higher per os virulence to Spodoptera litura larvae than BbV28 in standardized bioassays with normal conidia for both cuticle penetration and ingestion or heat-killed conidia for ingestion only. Conclusively, Phyd1-t1 is a useful tool for enhancing beneficial protein expression, such as vip3Aa1, in fungal conidia, which are the active ingredients of mycoinsecticides.

The Pathogen and Wound Induces Expression of Genes Related to Proanthocyanidins (PAs) Synthesis in Cotton Leaves

Responses to biotic and abiotic stress have been extensively studied in plants. In the current proteomic study, the cotton (Gossypium hirsutum L.) seedlings were infected with Verticillium dahliae by root-dip inoculation using suspension of fungal conidia. The different proteins were analyzed by two-dimensional gel elactrophoresis (2-DE), and flavanone 3-hydroxylase (F3H) showed a significantly up-regulation in cotton leaf after V. dahliae infection. Further research revealed F3H and the downstream genes of F3H in proanthocyanidins (PAs) biosynthesis were also significantly induced and showed coordinate expression patterns during wounding. The results indicate that PAs in cotton act an important role in response to infection V. dahliae and wounding.

Surface display of Gaussia princeps luciferase allows sensitive fungal pathogen detection during cutaneous aspergillosis

Non-invasive imaging techniques in microbial disease models have delivered valuable insights in the intimate pathogen-host interplay during infection. Here we describe evaluation and validation of a transgenic bioluminescence reporter strain of the human-pathogenic mold Aspergillus fumigatus, one of the main fungal pathogens affecting immunocompromised individuals. Expression and surface display of the Gaussia princeps luciferase allowed sensitive and rapid detection of luminescence emitted from this strain after substrate addition, with photon fluxes strongly correlating to the amounts of fungal conidia or germlings. The reporter strain allowed spatio-temporal monitoring of infection in a cutaneous model of aspergillosis, where neutropenic mice maintained the fungal burden while immunocompetent ones were able to clear it entirely. Most importantly, antifungal therapy could be followed in this type of disease model making use of the bioluminescent A. fumigatus strain. In conclusion, combining sensitivity of the Gaussia luciferase with a surface display expression system in the fungal host allows longitudinal infection studies on cutaneous forms of aspergillosis, providing perspective on drug screening approaches at high-throughput.

Gray Mold on Carrot Caused by Botrytis cinerea in Korea

Gray mold caused by Botrytis cinerea was found on a carrot seedling in a greenhouse and a field at Daegwallryeong, Gangwon Province in 2007-2009. Symptoms included irregular, brown, blight, or chlorotic halo on leaves and petioles of the carrots. Fungal conidia were globose to subglobose or ellipsoid, hyaline or pale brown, nonseptate, one celled, () in size, and were formed on botryose heads. B. cinerea colonies were hyaline on PDA, and then turned gray and later changed dark gray or brown when spores appeared. The fungal growth stopped at , temperature range for proper growth was on MEA and PDA. Carrots inoculated with ml conidial suspension were incubated in a moist chamber at for pathogenicity testing. Symptoms included irregular, brown, water-soaked rot on carrot roots and irregular, pale brown or dark brown, water-soaked rot on leaves. Symptoms were similar to the original symptoms under natural conditions. The pathogen was reisolated from diseased leaves, sliced roots, and whole roots after inoculation. As a result, this is the first report of carrot gray mold caused by B. cinerea in Korea.

First Report of Twig Blight Disease of Citrus Caused by Haematonectria haematococca in the Philippines.

Citrus has recently become one of the most important crops in the Philippines with approximately 151,000 t of production in 2005. A new disease affected citrus (Citrus reticulata Blanco cv. Satsuma) twigs and trunks in 2005. The problem has caused significant concerns to the industry because of its destruction and high severity of infection. Symptoms include twig drying, bark hardening, twig girdling, leaf chlorosis, and defoliation. Infection occurs at all stages of the plant where severe symptoms were observed from January to April. Average temperature of these months ranged from 20 to 28°C. Affected twigs were often covered with pink or salmon-colored fungal propagules (1). In April 2009, citrus twigs displaying the above-described symptoms and signs were collected from Malabing Valley, Kasibu, Nueva Vizcaya, the Philippines (16°20'4.6912″N, 121°16'41.0742″E). Fungal perithecia were surface sterilized and observed with a stereomicroscope. Single ascospore was picked and inoculated into the peptone-pentachloronitrobenzene medium (2) and the plates were incubated at 22 ± 2°C for 7 days with a 12-h light/dark regimen. Periodically, the plates were screened for the growth of mycelia or discrete colonies on the medium. The cultured fungus produced microconidia and multiple canoe-shaped macroconidia. With a hand-held sprayer, approximately 20 ml of distilled water containing fungal conidia (2 × 105 spores/ml) was inoculated each onto 1-year-old cv. Satsuma citrus (30 plants) bearing young twigs. Control plants were sprayed with sterile distilled water. After inoculation, plants were maintained at 20 to 28°C and 75 to 85% relative humidity, enclosed in clear plastic bags, placed under 70% woven shade cloth, and watered regularly. The inoculated plants started showing the initial twig blight symptoms in all inoculated branches at 37 days post inoculation (dpi). The occurrence of pink-to-orange perithecia of Haematonectria haematococca was observed at 45 dpi, which has similar morphological appearance to the perithecia collected from the field. Symptoms were not observed on branches treated with sterile water. H. haematococca was reisolated from the symptomatic twigs and the recovered isolates were morphologically similar to H. haematococca, thus completing Koch's postulates. Control plants remained healthy. DNA was extracted from all isolates, and the nuclear ribosomal internal transcribed spacer (ITS) region was amplified with primers ITS1 and ITS4 and sequenced. A portion of the ITS sequence has been deposited in the NCBI database (GenBank Accession No. HQ696788). A BLAST search of the NCBI database with the ITS sequence revealed H. haematococca (Berk. & Broome) Samuels & Nirenberg as the closest match with 100% sequence similarity. To our knowledge, this is the first report of H. haematococca causing citrus twig blight in the Philippines. To maintain the economic profitability of the citrus industry in the Philippines, control measures must be implemented to minimize tree loss.

Monocyte-Derived Dendritic Cells Pulsed with Aspergillus Fumigatus Components Enhance the Anti-Aspergillus Effect of Human Natural Killer Cells

Abstract 2983Dendritic cells (DCs) are well characterized for their ability to induce adaptive immune responses. To mimic these properties, in vitro generated monocyte-derived DCs (moDCs) have been extensively studied and used as antigen presenting cells, initiating specific T cell immune responses against virus infected and malignant cells. There is increasing evidence that the immunoregulatory effect of DCs is exerted also towards innate immune effector cells, like NK cells. Recently, we have shown a significant anti-Aspergillus fumigatus impact of NK cells, mainly mediated by the release of IFN- γ, which demonstrated direct fungicidal properties. The interest for our study is driven by the high mortality characterizing invasive aspergillosis, an infection of immunosuppressed patients caused mainly by this fungus. To date, it has been shown that NK cells activated by DCs demonstrate increased IFN- γ release, proliferation and cytotoxicity against malignant cells. In our study, we have investigated the potential effect of moDCs, previously pulsed with fungal proteins and with cell wall fragments on NK cells and on their cytotoxicity against A. fumigatus.For the generation of moDCs, we used CD14+ cells isolated from peripheral blood of healthy donors by positive selection with magnetic microbeads. Cells were cultured for 5 days in serum-free GMP grade medium supplemented with GM-CSF and IL-4. On the second day of culture, immature DCs were pulsed either with a β-(1, 3)- glucan–rich alkali-insoluble (AI) cell wall fraction extracted from fungal conidia or from mycelia, inactivated conidia and germ tubes or with the recombinant catalase Cat1 and transglycosidase Crf1. Three hours later, DCs were maturated with TNF- α. Before being used, DC properties were characterized by quantifying the expression of CD1a, CD14, CD40, CD80, CD83, CD86, HLA-I, HLA-DR, CCR7, Dectin-1, TLR-2, TLR-4, IL12p35, IL15, and IFN- γ. Autologous NK cells were obtained by magnetic negative selection of peripheral mononuclear cells and were kept frozen in human serum supplemented with 8% DMSO at −80°C. DC/ NK cell co-cultures were set in unconditioned GMP grade medium on different ratios (1:1, 1:5, 1:10). To evaluate proliferation, NK cells were stained with CFSE. Proliferation was measured at the 7th day of DC / NK co-culture. IFN- γ release by NK cells was quantified by FACS analysis after 16 h of DC / NK co-culture. Finally, the antifungal impact on A. fumigatus germ tubes was evaluated by 2, 3-bis[2-methoxy-4-nitro-5-sulpho-phenyl]2H-tetrazolium-5-carboxanilide (XTT) assays.DCs pulsed with the different fungal stimuli demonstrated a ‘mature' surface profile with negative CD1a & CD14 expression and high positivity of CD40, CD80, CD83, CD86, HLA-I, HLA-DR & CCR7. All DCs produced IL12p35 and IL15, but no IFN- γ. The expression of Dectin-1 was strongly down-regulated (p<.001) by the alkali-insoluble cell wall fraction and by germlings, moderately by conidia (p<.05) and it was unaffected after Cat1 and Crf1 stimulation. This observation suggests a major involvement of Dectin-1 and its ligand, β-(1, 3)-glucan in the interaction of moDCs with A. fumigatus. In contrast, TLR-2 and TLR-4 were not found to be regulated on DCs. Furthermore, DCs induced the proliferation of NK cells, higher DC / NK ratios elicited higher NK cell expansion. DCs stimulated with AI cell wall fractions or germlings provoked a significantly higher proliferation of NK cells in comparison to unstimulated DCs (p<.05). Similarly, significant up-regulation of IFN- γ expression was observed in NK cells co-cultured with DCs previously pulsed with germ tubes and with fungal cell wall fractions, respectively (p<.05). Finally, we revealed significant higher fungal damage in NK cell / DC co-cultures (especially in those primed with fungal cell wall fractions or germlings), compared to NK cell or DC cultures alone (p<.05).To summarize, our study shows for the first time that moDCs pulsed with A. fumigatus cell wall components induce NK cell proliferation and potentiate IFN- γ release and cytotoxicity against this fungus. These results provide evidence for a role of moDCs as activators of NK cells towards an immune response against IA. Disclosures:No relevant conflicts of interest to declare.

Proteasome Inhibitors Affect Appressorium Formation and Pathogenicity of the Rice Blast Fungus, Magnaporthe oryzae

Previously, we identified the 20S proteasome <TEX>${\alpha}$</TEX>-subunit of Magnaporthe oryzae (M. oryzae) induced during appressorium formation, and detected an increase in multiple protein ubiquitination during the early appressorium formation process (Kim et al., 2004). In this study, we further attempted to determine whether the proteasome is involved in the appressorium formation of M. oryzae both in vitro and in planta, using proteasome inhibitors. A significant increase in 20S proteasome during fungal germination and appressorium formation was observed using Western blot analysis with 20S proteasome antibody, demonstrating that proteasome-mediated protein degradation was involved in appressorium formation. Pharmacological analysis using proteasome inhibitors, MG-132, proteasome inhibitor I (PI) and proteasome inhibitor II (PII) revealed that germination and appressorium formation were delayed for 4 to 6 h on rice leaf wax-coated plates. Similarly, the treatment of proteasome inhibitors with fungal conidia on the rice leaf surface delayed appressorium formation and host infection processes as well. Additionally, fungal pathogenicity was strongly reduced at 4 days' postfungal infection. These data indicated that the fungal 20S proteasome might be involved in the pathogenicity of M. oryzae by the suppression of germination and appressorium formation.

LongSAGE gene-expression profiling of Botrytis cinerea germination suppressed by resveratrol, the major grapevine phytoalexin

The ascomycetes Botrytis cinerea is one of the most studied necrotrophic phytopathogens and one of the main fungal parasites of grapevine. As a defense mechanism, grapevine produces a phytoalexin compound, resveratrol, which inhibits germination of the fungal conidium before it can penetrate the plant barriers and lead to host cell necrotrophy. To elucidate the effect of resveratrol on transcriptional regulation in B. cinerea germlings, two LongSAGE (long serial analysis of gene expression) libraries were generated in vitro for gene-expression profiling: 41 428 tags and among them, 15 665 unitags were obtained from resveratrol-treated B. cinerea germlings and 41 358 tags, among them, 16 362 unitags were obtained from non-treated B. cinerea germlings. In-silico analysis showed that about half of these unitags match known genes in the complete B. cinerea genome sequence. Comparison of unitag frequencies between libraries highlighted 110 genes that were transcriptionally regulated in the presence of resveratrol: 53 and 57 genes were significantly down- and upregulated, respectively. Manual curation of their putative functional categories showed that primary metabolism of germinating conidia appears to be markedly affected under resveratrol treatment, along with changes in other putative metabolic pathways, such as resveratrol detoxification and virulence-effector secretion, in B. cinerea germlings. We propose a hypothetical model of cross talk between B. cinerea germinating conidia and resveratrol-producing grapevine at the very early steps of infection.

Dissemination of entomopathogenic fungi using Busseola fusca male as vector

The stem borer, Busseola fusca (Fuller), is an important pest of maize Zea mays L. and sorghum Sorghum bicolor (L.) in eastern and southern Africa. To control this pest, biological control methods including the use of entomopathogenic fungi are being considered. The pathogenicity of one isolate of Metarhizium anisopliae (Metsch.) Sorok. and one isolate of Beauveria bassiana Bals. (Vuill.) were first tested on different developmental stages of B. fusca including eggs, neonate, 2nd and 3rd-instar larvae. Both fungal isolates were pathogenic to all the stages tested. However, differences in mortality were observed among larvae that hatched from treated egg masses. Experiments were conducted thereafter to test whether B. fusca males could serve as a vector for fungal conidia to contaminate B. fusca females and subsequently eggs and larvae. Results demonstrated that B. fusca males successfully transferred inoculum to females during copulation, which in turn transmitted it to the eggs they laid on maize plants, resulting in the decrease of leaf damages.

A biotechnology for the production of new biodiesel fuel

A new biotechnology for the production of biodiesel fuel on the basis of the lipids of filamentous fungi has been developed. The oleaginous mucoralean fungus, which produces up to 47.0–50.0% of lipids similar to rapeseed oil in their composition, are used as a producer. The duration of the fermentation process at 27–28°C is 4–5 days. The main characteristics of the biodiesel fuel based on fungal lipids satisfy the requirements of the basic standards, in particular, EN14214. The possibilities for creating a wasteless closedcycle technology based on the production of biodiesel and chitin in a single fermentation, applying stimulators for the germination of fungal conidia to reduce the duration of fermentations, and using glycerin formed in the synthesis of methyl esters as a source of carbon in a fermentation medium, along with wood hydrolyzates and the wastes of certain food plants in the fermentation process, are shown.

Evaluation of inhibitory effects of citric and tartaric acids and their combination on the growth of Trichophyton mentagrophytes, Aspergillus fumigatus, Candida albicans, and Malassezia furfur

The present study was conducted to evaluate the effects of citric and tartaric acids and their combination on the growth inhibition of some important pathogenic fungi in vitro. Trichophyton mentagrophytes var. mentagrophytes, Candida albicans, Aspergillus fumigatus, and Malassezia furfur were cultured into specific media, and subsequently, fungal conidia were harvested from the medium surface and counted by hemacytometer method. The antifungal susceptibility test of citric and tartaric acids and their combination against fungi were assayed by broth macrodilution technique. The results demonstrated that citric acid had more fungistatic and fungicidal activities than those of tartaric acid against all pathogenic fungi tested, and its effect on filamentous fungi was higher than that on the yeasts. Antifungal activity of the acid combination was similar to citric acid but higher than tartaric acid alone. Further research is needed to assess the efficacy of citric and tartaric acids as inhibitors of fungal growth in clinical trials, especially in treatment of patients with fungal infections.

First Report of Anthracnose Caused by Glomerella acutata on Chili Pepper in China.

A new anthracnose disease on chili pepper (cayenne pepper cv. Hongxiu 2003, fruiting type pepper) was found in Zhijiang County, Hunan, China in 2009. The disease was observed only on the fruits. Lesions were generally elongated, on which dark acervuli were arranged concentrically. Later, cracking of older lesions was observed. With a microscope, fungal conidia were observed to be 15.8 × 4.1 μm, fusiform or oval with one end acute, and single celled with two to seven oil globules. No setae were found on the acervuli. Eight isolates (HNZJ001-HNZJ008) showed no difference in colony feature when cultured on potato dextrose agar. All the isolates showed white growth at the early stages, but colonies turned pink when they produced powdery spores and then finally became red gray. The average colony diameter was 68.5 to 72.3 mm after 7 days with obvious gray black concentric rings because of the development of aerial and substrate mycelia. After a needle-prick inoculation with a suspension of 1 × 106 spores per ml of HNZJ001 on 30 chili pepper fruits with three repeats, the same symptoms were observed and the same fungus was recovered. In bioassays, HNZJ001 caused lesions on both mature and immature fruits, while Glomerella cingulata strain LSQ1 (GenBank Accession No. HQ607386) used as a control did not infect immature fruits. PCR amplification was carried out by utilizing universal rDNA-ITS primer pair ITS4/ITS5. Sequencing of the PCR products of HNZJ001 (GenBank Accession No. GU059863) showed 100% identity to G. acutata (GenBank Accession No. EU008863) and Colletotrichum acutatum (GenBank Accession No. AF207794) after a BLAST search. The pathogen was identified as G. acutata (asexual stage: C. acutatum) on the basis of morphological characters and rDNA-ITS sequence analysis. Worldwide, it has been reported that pepper anthracnose might be caused by up to five species of Glomerella (Colletotrichum): G. cingulata, C. coccodes, C. capsici, C. dematium, and G. acutata (2), among which only the first three were previously reported in China. In recent years, G. acutata was reported on such plants as apple (3) and strawberry (1) in China, but not on pepper. To our knowledge, this is the first report of G. acutata on chili pepper in China.

Influence of Fungal Odor on Grooming Behavior of the Termite,Coptotermes formosanus

The termite Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae) protects itself from entomopathogenic fungus by mutual grooming behavior. C. formosanus removes foreign organisms, such as fungal conidia, from the body surface of its nestmates by mutual grooming behavior and eating them. The conidia removal rate from the body surface differed according to the isolate of entomopathogenic fungi (Beauveria brongniartii 782, Paecilomyces fumosoroseus K3, and Metarhizium anisopliae 455), and the removal rate of the fungal isolates seemed to depend on feeding preference, which was detrmined using paper discs moistened with a fungal suspension. In addition, it was found that C. formosanus without antennae groomed their nestmates more frequently than those with antennae. Consequently, it seems that C. formosanus antennae detect substances without touching, such as via odor, and it affects the efficiency of grooming behavior. The results of single sensillum recording support the hypothesis that C. formosanus are capable of distinguishing three species of fungi by their odors.

Integration of insecticidal protein Vip3Aa1 into Beauveria bassiana enhances fungal virulence to Spodoptera litura larvae by cuticle and per Os infection.

The entomopathogenic fungus Beauveria bassiana acts slowly on insect pests through cuticle infection. Vegetative insecticidal proteins (Vip3A) of Bacillus thuringiensis kill lepidopteran pests rapidly, via per os infection, but their use for pest control is restricted to integration into transgenic plants. A transgenic B. bassiana strain (BbV28) expressing Vip3Aa1 (a Vip3A toxin) was thus created to infect the larvae of the oriental leafworm moth Spodoptera litura through conidial ingestion and cuticle adhesion. Vip3Aa1 ( approximately 88 kDa) was highly expressed in the conidial cytoplasm of BbV28 and was detected as a digested form ( approximately 62 kDa) in the larval midgut 18 and 36 h after conidial ingestion. The median lethal concentration (LC(50)) of BbV28 against the second-instar larvae feeding on cabbage leaves sprayed with conidial suspensions was 26.2-fold lower than that of the wild-type strain on day 3 and 1.1-fold lower on day 7. The same sprays applied to both larvae and leaves for their feeding reduced the LC(50) of the transformant 17.2- and 1.3-fold on days 3 and 7, respectively. Median lethal times (LT(50)s) of BbV28 were shortened by 23 to 35%, declining with conidial concentrations. The larvae infected by ingestion of BbV28 conidia showed typical symptoms of Vip3A action, i.e., shrinkage and palsy. However, neither LC(50) nor LT(50) trends differed between BbV28 and its parental strain if the infection occurred through the cuticle only. Our findings indicate that fungal conidia can be used as vectors for spreading the highly insecticidal Vip3A protein for control of foliage feeders such as S. litura.

Synthesis and in vitro and in vivo Antifungal Activity of the Hydroxy Metabolites of Saperconazole

Herein we describe the scalable diastereoselective and enantioselective syntheses of eight enantiomers of hydroxy metabolites of saperconazole. The in vitro antifungal activity of the eight stereoisomers (compounds 1-8) was compared against a broad panel of Candida spp. (n=93), Aspergillus spp. (n=10), Cryptococcus spp. (n=19), and dermatophytes (n=27). The four 2S isomers 1-4 of the new agent were generally slightly more active than the four 2R isomers 5-8. All eight isomers were tested in a model of experimental A. fumigatus infection in guinea pigs by intravenous inoculation of the fungal conidia. Treatment doses were 1.25 mg kg(-1) and 2.5 mg kg(-1) per day. Infection severity was measured in terms of mean survival time (MST) after infection and mean tissue burdens in brain, liver, spleen, and kidney at postmortem examination. Among the eight isomers, the 2S diastereomers 1-4 showed a generally higher level of activity than the 2R diastereomers 5-8, revealing compounds 1 and 4 as the most potent overall in eradicating tissue burden and MST. Compared with reference compounds itraconazole and saperconazole, the hydroxy isomers 1-8 are less potent inhibitors of the growth of A. fumigatus in vitro and of ergosterol biosynthesis in both A. fumigatus and C. albicans.

Histopathology Caused by the Entomopathogenic Fungi,Beauveria bassianaandMetarhizium anisopliae, in the Adult Planthopper,Peregrinus maidis, a Maize Virus Vector

The planthopper Peregrinus maidis (Ashmead) (Hemiptera: Delphacidae) is an important vector of maize viruses in tropical and subtropical areas. Planthoppers are biologically controlled with several species of entomopathogenic fungi that have been isolated from these insect pests of rice in Asia. Beauveria bassiana (Balsamo-Crivelli) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae) appear to be the most useful against planthoppers because of their ease of mass production, storage, virulence, and application. In the present study, adults of P. maidis infected with B. bassiana and M. anisopliae were observed under light and scanning electron microscopy to characterize morphologically the process of infection and the development of these fungi, prior to and after the death of the host. The hydrophobic conidia of both fungal species were able to attach to all body regions, with a preference for surfaces containing hairs. Few germinated conidia were observed on the insect's body surface at 24, 48, and 72 hr post-inoculation. On the cuticular surface of P. maidis treated with B. bassiana and M. anisopliae, bacillus-like bacteria were observed. These microorganisms could be interacting with fungal conidia, playing a role of antibiosis that will not allow the fungal pathogens to germinate and penetrate. In the colonization events observed in this study, the formation and multiplication of hyphal bodies by both fungal species inside the host's body was noted. The host's whole body was invaded by hyphae between five and six days post-inoculation, and body fat was the most affected tissue.

Performance of a Metarhizium anisopliae-treated semiochemical-baited trap in reducing Amblyomma variegatum populations in the field

Experiments were carried out to evaluate the efficacy of Metarhizium anisopliae (Metsch.) Sorok. (Ascomycota: Hypocreales)-treated semiochemical-baited traps for control of Amblyomma variegatum Fabriscius (Acari: Ixodidae) under field conditions. Unfed A. variegatum adults (118) were seeded in each 100-m plot and allowed to acclimatise for 3 days. On the fourth day (Day 4), an emulsifiable formulation of M. anisopliae (consisting of 49.5% sterile distilled water, fungal conidia, 49.5% corn oil and 1% Tween 80) titrated at 10 9 conidia ml −1 was applied in semiochemical-baited traps (900 cm 2) which were placed at five spots within the plot. The control and fungal treatments were repeated after 14 and 28 days soon after rotating the traps clockwise (45°) in order to cover different sections of the plot. In the control plots, emulsifiable formulation without fungus was applied in the semiochemical-baited traps. Six weeks after the initiation of the experiments, five semiochemical-baited traps (untreated) were deployed in each plot for 3 successive days to trap ticks in the treated and control plots. The percentage of ticks recovered in the fungus-treated plots were significantly lower (31.1 ± 5.2%) than in the control plots (85.6 ± 3%) ( P < 0.001), which represented a relative tick reduction of 63.7%. Mortality of 93.8 ± 2.3% was observed among the ticks that were recovered from the field and maintained in the laboratory for 2 weeks; while only 3.3 ± 0.9% died from the control plots. The results of this study open up the possibility of developing an environmentally friendly and low cost application strategy to control Amblyomma ticks.