Biological Control of Celery Powdery Mildew Disease Caused by Erysiphe heraclei DC In Vitro and In Vivo Conditions

The present study aimed to investigate the potentiality of certain biocontrol agents, namely Bacillus subtilis, B. pumilus, B. megaterium, Pseudomonas fluorescens, Serratia marcescens, Trichoderma album, T. harzianum and T. viride, as well as the synthetic fungicide difenoconazole to control celery powdery mildew caused by Erysiphe heraclei DC, in vitro (against conidia germination and germ tube length of E. heraclei) and in vivo (against disease severity and AUDPC). In vitro, it was found that the antifungal activity of the tested biocontrol agents significantly reduced the germination percentage of the conidia and germ tube length of the pathogen. The reduction in conidia germination ranged between 88.2% and 59.6% as a result of the treatment with B. subtilis and T. album, respectively compared with 97.1% by the synthetic fungicide difenoconazole. Moreover, the fungicide achieved the highest reduction in germ tube length (92.5%) followed by B. megaterium (82.0%), while T. album was the least effective (62.8%). Spraying celery plants with the tested biocontrol agents in the greenhouse significantly reduced powdery mildew severity, as well as the area under the disease progress curve (AUDPC), after 7, 14, 21 and 28 days of application. In this regard, B. subtilis was the most efficient followed by B. pumilus, S. marcescens and B. megaterium, with 80.1, 74.4, 73.2 and 70.5% reductions in disease severity, respectively. In AUDPC, reductions of those microorganisms were 285.3, 380.9, 396.7 and 431.8, respectively, compared to 1539.1 in the control treatment. On the other hand, the fungicide difenoconazole achieved maximum efficacy in reducing disease severity (84.7%) and lowest AUDPC (219.3) compared to the other treatments. In the field, all the applied biocontrol agents showed high efficiency in suppressing powdery mildew on celery plants, with a significant improvement in growth and yield characteristics. In addition, they caused an increase in the concentration of leaf pigments, and the activities of defense-related enzymes such as peroxidase (PO) and polyphenol oxidase (PPO) and total phenol content (TPC). In conclusion, the results showed the possibility of using tested biocontrol agents as eco-friendly alternatives to protect celery plants against powdery mildew.

Glucosylceramide Plays a Role in Fungal Germination, Lipid Raft Organization and Biofilm Adhesion of the Pathogenic Fungus Scedosporium aurantiacum

Infections caused by Scedosporium species present a wide range of clinical manifestations, from superficial to disseminated, especially in immunocompromised patients. Glucosylceramides (GlcCer) are glycosphingolipids found on the fungal cell surface and play an important role in growth and pathogenicity processes in different fungi. The present study aimed to evaluate the structure of GlcCer and its role during growth in two S. aurantiacum isolates. Purified GlcCer from both isolates were obtained and its chemical structure identified by mass spectrometry. Using ELISA and immunofluorescence techniques it was observed that germination and NaOH-treatment of conidia favor GlcCer exposure. Monoclonal anti-GlcCer antibody reduced germination when cultivated with the inhibitor of melanin synthesis tricyclazole and also reduced germ tube length of conidia, both cultivated or not with tricyclazole. It was also demonstrated that anti-GlcCer altered lipid rafts organization, as shown by using the fluorescent stain filipin, but did not affect the susceptibility of the cell surface to damaging agents. Anti-GlcCer reduced total biomass and viability in biofilms formed on polystyrene plates. In the presence of anti-GlcCer, germinated S. aurantiacum conidia and biofilms could not adhere to polystyrene with the same efficacy as control cells. These results highlight the relevance of GlcCer in growth processes of S. aurantiacum.

Selection of Beauveria bassiana (Hypocreales: Cordycipitaceae) strains to control Xyleborus affinis (Curculionidae: Scolytinae) females

Background Xyleborus affinis Eichhoff (Coleoptera: Curculionidae) is an ambrosia beetle reported to affect avocado trees (Persea americana Mill.). The use of the entomopathogenic fungus (EPF) Beauveria bassiana (Bals.-Criv.) Vuill. for ambrosia beetle control represents an alternative to insecticides. Methods This study was designed in two stages to select B. bassiana strains with potential to control X. affinis females. In the first stage, 19 B. bassiana Mexican strains from EPF collection, isolated from Coleoptera (CHE-CNRCB, http://www.gob.mx/senasica/documentos/coleccion-de-hongos-entomopatogenos), were tested. Analyses included radial growth rate, conidial yield, spore germination, and germ tube length. Results were analysed by Principal Component Analysis (PCA) to identify clusters within favourable growth phenotypes. For the second stage, 10 selected strains were re-analysed for virulence-related metabolic characteristic, including cell wall-bound cuticle-degrading enzymes–Pr1-like proteases and β-N-acetyl glucosaminidases (NAGase) chitinases, conidial hydrophobicity and monopolar germination parameters. A second PCA analysis was run for those virulence parameters analysed, and upon results strains CHE-CNRCB 44, 171, 431 and 485 were selected and tested against X. affinis females. Females were treated with a 1 × 108 conidia mL−1 suspension (recommended rate), using a Potter Tower. Results All strains showed insecticidal activity, inducing up to 58% mortality; about 30% dead beetles developed aerial mycelia (CHE-CNRCB 485) and the fastest mortality rate was t0 = 1.95 (CHE-CNRCB 44). Conclusion Since all selected strains showed virulence against X. affinis females, results indicated the possibility of selecting B. bassiana strains based on multiple metabolic attributes, as a preliminary test to perform bioassays against order-related target insects.

Effect of Legume Extracts on Germination, Seedling Health of Beans (Phaseolus vulgaris L.) and Soil Microorganisms

Application of undecomposed green manure has been reported to cause poor emergence and establishment of common beans in the field. Therefore, to understand the mechanisms’ contributing to the poor crop establishment, the effect of extracts from fresh and decomposed legume green manures on bean seed germination, fungal mycelial growth, spore germination and germ tube elongation were evaluated. The extracts were prepared in either ethanol or distilled water. Data was collected on percentage seed germination, seedling length, mycelial radial growth, spore germination and germ tube elongation. Ethanol extracts from fresh lablab inhibited bean germination by 56%, increased mean germination time to 8 days, and decreased germination index while ethanol extracts of groundnut and beans caused highest inhibition in bean shoot length and reduced biomass. Ethanol extracts from fresh green manures significantly inhibited fungal mycelial growth while the aqueous extracts from beans, groundnuts and soybean had significant level of antifungal activity while aqueous lablab extracts stimulated mycelial. Aqueous extract of lablab and soybean enhanced spore germination by over 70% with more pronounced effect on germ tube length and number of germ tubes by 8.0% and 13% respectively. The study comparatively reveals that the extract of lablab was inhibitory to common bean germination compared to other legume extracts and also stimulated the growth of root rot pathogens that may have resulted in poor establishment of beans.

Effects of Sporidiobolus pararoseus Y16 on Postharvest Blue Mold Decay and the Defense Response of Apples

The efficacy of Sporidiobolus pararoseus Y16 in controlling postharvest blue mold caused by Penicillium expansum on apples and the defense response involved were evaluated. The results suggested that the decay incidence of blue mold of apples treated by S. pararoseus Y16 was significantly reduced compared with the control. In vitro testing indicated that germination of spores and germ tube length of P. expansum were markedly inhibited by S. pararoseus Y16. Meanwhile, polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), and catalase (CAT) activities and several pathogenesis-related (PR) gene expression levels (including PR3, PR4, PR5, and PR9) were determined. In apples, the activities of PPO, POD, CAT, and PAL were significantly induced by S. pararoseus Y16 treatment compared with the control fruits. The relative expression levels of PR3 and PR4 were significantly induced at 4 and 6 d, while PR5 was significantly induced at 4 and 6 d and PR9 was significantly induced at 4 d. Therefore, the reduction in apple fruit decay by S. pararoseus Y16 treatment could be related to the increased activities of related enzymes and proteins involved in the defense against pathogens, which suggest that S. pararoseus Y16 is a potential antagonistic yeast.

In vitro effect of substrate, temperature and photoperiod on Phakopsora pachyrhizi urediniospore germination and germ tube growth

In vitro experiments were conducted to assess the effects of substrate, temperature and time of exposure to temperature and photoperiod on P. pachyrhizi uredospore germination and germ tube growth. The following substrates were tested: water-agar and soybean leaf extract-agar at different leaf concentrations (0.5, 1.0, 2.0 and 4.0 g of leaves and 15g agar/L water), temperatures (10, 15, 20, 25, 30, and 35oC) and times of exposure (1, 2, 3, 4, 5, 6, 7, and 8 hours) to temperature and 12 different photoperiods. The highest germination and germ tube length was found for the soybean leaf extract agar. Maximum P. pachyrhizi uredospore germination was obtained at 21.8 and 22.3°C, and maximum germ tube growth at 21.4 and 22.1°C. The maximum uredospore germination was found at 6.4 hours exposure, while the maximum germ tube length was obtained at 7.7 h exposure. Regarding photoperiod, the maximum spore germination and the maximum uredospore germ tube length were found in the dark. Neither spore germination nor uredospore germ tube growth was completely inhibited by the exposure to continuous light.

Effect of temperature, relative humidity and light on conidial germination of oak powdery mildew (Microsphaera alphitoides Griff. et Maubl.) under controlled conditions

The influence of temperature, humidity and light on the conidial germination and germ tube elongation of oak powdery mildew (Microsphaera alphitoides Griff. et Maubl.) was studied in controlled conditions. The maximal germ tube length was attained at 25?C, whereas at lower and higher than optimal temperatures, germ tube growth was significantly lower. Germ tubes begin to develop at all values of relative humidity (10-100%), reaching the maximum length at 90%. The development of germ tubes was the most intense in full light and the lowest in total darkness. The artificial infection of floating leaves showed that an increasing age had an inhibitory effect on the mycelium development and spore formation. Since conidia play a crucial role in powdery mildew epidemiology, it is of particular importance to elucidate the influence of environmental factors in the complex relations that exist between the plant and its pathogen.

Influence of Iron on Cylindrocarpon Root Rot Development on Ginseng

Cylindrocarpon root rot, caused by Cylindrocarpon destructans, is an important disease on ginseng (Panax quinquefolius) in Canada. We studied the effects of iron (Fe) on disease severity and pathogen growth. When Hoagland's solution was amended with Fe at 56 and 112 μg/ml compared with 0 μg/ml, disease initiation and final severity on hydroponically maintained ginseng roots was significantly (P<0.0001) enhanced. Under field conditions, wounding of roots with a fine needle followed by application of 0.05% FeNaEDTA to the rhizosphere of treated plants significantly enhanced Cylindrocarpon root rot in 2003 and 2004 compared with unwounded roots with Fe or wounded roots without Fe. Foliar applications of Fe (as FeNaEDTA) to ginseng plants three times during the 2002 and 2003 growing seasons significantly increased Fe levels in root tissues. These roots developed larger lesions following inoculation with C. destructans in vitro. When radioactive Fe (59Fe) was applied to the foliage of ginseng plants, it was detected in the secondary phloem and in cortical and epidermal tissues within 1 week. Artificially wounded areas on the roots accumulated more 59Fe than healthy areas. Diseased tissue also had threefold higher levels of phenolic compounds and Fe compared with adjoining healthy tissues. High-performance liquid chromatography analysis revealed enhanced levels of protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, cinnamic acid, phloridizin, and quercetin. Phenolic compounds produced in diseased and wounded tissues sequestered Fe in vitro. The effects of Fe on mycelial growth, conidial germ tube length, and secondary branching of germ tubes of C. destructans were examined in vitro. When grown on Chrome-azurol S medium, Fe also was sequestered by C. destructans through siderophore production, which was visualized as a clearing pigmented zone at the margin of colonies. Mycelial dry weight was significantly increased in glucose/ yeast broth containing Fe at 56 or 112 μg/ml. Conidial germ tube length and secondary branching of hyphae also were enhanced after 8 and 16 h by Fe. Colony growth of C. destructans was not enhanced by Fe, but significantly greater spore production was observed with Fe at 56 and 112 μg/ml compared with no Fe in the medium. Although these levels of Fe had no effect on fungal pectinase enzyme activity, polyphenoloxidase (PPO) activity was significantly (P <0.0001) enhanced. We conclude that Fe enhances Cylindrocarpon root rot through enhanced pathogen growth, sporulation, and PPO enzyme activity. Fe sequestered by phenolic compounds produced in wounded tissues can enhance Fe levels at the site of infection. The pathogen also has the ability to sequester Fe at these sites.

The effect of light and temperature on in vitro germination and germ tube growth of urediniospores of Tranzschelia discolor

The effect of light on in vitro germination of urediniospores of Tranzschelia discolor was studied over time at different intensitities (up to 400 8E m-2 s-l) within the temperature range 5�C to 20�C. A model was also developed from the data to predict germination at different combinations of light, temperature and times of leaf wetness. Light retarded the germination process, and its effect increased in direct proportion to intensity. At 20�C, for example, the time taken to exceed 80% germination increased from 2 h in the dark to 9 h at 200 8E m-2 s-l. The model showed that there was an interaction between light and temperature, with the effect of light becoming more pronounced as the temperature declined below 20�C. Germination percentages of the order of 90% were, however, recorded within 24 h at all combinations of light intensity and temperature studied. Light also influenced germ tube growth, causing a reduction in the rate of growth. As in germination, its effect increased with increasing light intensity. At 20�C, the average germ tube length at 9 h was 541 8m in the dark, compared with 227 8m at 200 8E m-2 s-1 and 148 8m at 400 8E m-2 s-l. A similar effect was observed at 5�C, where the average germ tube length at 24 h was 274 8m in the dark compared with 157 8m at 200 8E m-2 s-l. The effects of light on the germination and germ tube growth of urediniospores under field conditions are discussed.