Pathogenicity of Aschersonia spp. against whiteflies Bemisia argentifolii and Trialeurodes vaporariorum

Entomopathogenic fungi of the genus Aschersonia are specific for whitefly and scale insects. They can be used as biological control agents against silverleaf whitefly, Bemisia argentifolii and greenhouse whitefly, Trialeurodes vaporariorum. Forty-four isolates of Aschersonia spp. were tested for their ability to sporulate and germinate on semi-artificial media and to infect insect hosts. Seven isolates sporulated poorly (less than 1x10(7) conidia/dry weight) and 10 were not able to infect either of the whitefly species. Several isolates were able to produce capilliconidia. Infection level was not correlated with germination on water agar. After a selection based on spore production and infection, virulence of 31 isolates was evaluated on third instar nymphs of both whitefly species on poinsettia (Euphorbia pulcherrima). Whitefly infection levels varied between 2 and 70%, and infection percentages of B. argentifolii correlated with that of T. vaporariorum. However, mortality was higher for T. vaporariorum than for B. argentifolii, as a result of a higher 'mortality due to unknown causes.' Several isolates, among which unidentified species of Aschersonia originating from Thailand and Malaysia, A. aleyrodis from Colombia, and A. placenta from India showed high spore production on semi-artificial medium and high infection levels of both whitefly species.     

Effect of temperature and water activity on in vitro germination of Monilinia spp.

Aims:  This study evaluated the effect of temperature (0–38°C) and water activity ( a _w: 0·87–0·99) on the lag phase prior to germination and the percentage of germination over time for Monilinia laxa , Monilinia fructicola and Monilinia fructigena .
Methods and Results:  More than 80% of viable conidia germinated at 25°C and 0·99 a _w within 2 h for M. fructicola and M. fructigena and 4 h for M. laxa . There was no germination at 38°C, and all three Monilinia spp. germinated at 0°C. At the lowest a _w (0·87), none of the Monilinia spp. was able to germinate at any of the incubation temperatures studied. Whereas at 0·90 a _w, conidia were only able to germinate at 15, 25 and 30°C for the three species studied, except for M. fructicola at 15°C. In contrast, at 0·95, 0·97 and 0·99 a _w, germination occurred at all studied temperatures less 38°C. Generally, the lag phase was longer at low levels of a _w (0·90–095), and differences were more evident as temperatures were far from the optimum (0–5°C).
Conclusions:  Germination and lag phase period were markedly influenced by temperature and a _w, and in general when conditions of temperature and a _w were suboptimal, the lag phase was longer and the percentage of germination was lower.
Significance and Impact of the Study:  Knowledge of the germination requirements of this fungus is important in order to understand their behaviour in natural situations and to provide baseline data required for the construction of new prediction models. Our study might be used to develop a predictive model to understand and control the disease caused by Monilinia spp.     

Effect of fungicides on growth,germination and cuticle-degrading enzyme production by Lecanicillium muscarium

Lecanicillium muscarium Zare and Gams (previously known as Verticillium lecanii) is a well-known pathogen of arthropods. The influence of six fungicides on growth, sporulation, conidial germination and cuticle-degrading enzyme production by L. muscarium was investigated under laboratory conditions. The maximum reduction in vegetative growth, sporulation and conidial germination in relation to the control treatment was observed for propiconazole, whereas mancozeb and chlorothalonil caused the lowest reduction in these parameters. Propiconazole and pyraclostrobin caused higher reduction in enzyme activities (chitinase, Pr1, Pr2 and lipase) at all three concentrations (10, 100 and 1000 µg/ml), whereas low reduction in enzyme activities was caused by chlorothalonil and mancozeb when used at 10 µg/ml. The data presented can be used for future recommendations of these fungicides in Integrated Pest Management (IPM) programmes where L. muscarium is an important control agent.     

Effects of nutrients on germination of Verticillium lecanii (=Lecanicillium sp.) conidia and infection of greenhouse whitefly, (Trialeurodes vaporariorum)

Laboratory experiments were conducted to assess effects of nutrients on germination of Verticillium lecanii (=Lecanicillium sp.) conidia and infection of the greenhouse whitefly, Trialeurodes vaporariorum. Suspensions of V. lecanii conidia were prepared in four nutrient solutions: 2% glucose, 2% sucrose, 2% maltose, and 2% peptone. Suspensions in de-mineralized water served as the control. At 23°C the germination rate was highest in the 2% glucose solution, followed by sucrose, maltose, demineralized water, and peptone, respectively. Germ tube growth was greatest at 23°C in the 2% glucose solution after 10 h incubation. Results of the bioassays indicated that the nutrients influenced whitefly infection. Infection levels were highest for conidial suspensions (1×10⁶ conidia/mL) prepared in 2% glucose, and were significantly greater than for peptone, demineralized water and maltose. Infection levels at 1×10⁸ conidia/mL were not significantly different from each other for all materials tested. The potential use of nutrients in a spray formulation as a means of enhancing field efficacy are discussed.     

Prediction of conidial germination of Penicillium chrysogenum as influenced by temperature, water activity and pH

AIMS: Conidial germination of Penicillium chrysogenum was carried out under operating conditions compatible with a pastries manufacturing process. METHODS AND RESULTS: A range, limited by two experimental values, was defined for each environmental factor tested: temperature (15 or 25 degrees C), water activity (0.75 or 0.85) and pH (3.5 or 5.5). A closed device was made, which maintained an equilibrium between water activity of the culture medium and atmospheric relative humidity during 25 days, to follow spore germination. The combined effects of temperature, water activity and pH on spore germination were studied by applying factorial design methodology. CONCLUSIONS: Higher rates of spore germination were associated with a high level of water activity. The incubation temperature also had a positive effect. A significant positive interaction between water activity and temperature was observed. Under these specific experimental conditions, pH did not have a significant effect on conidial germination. SIGNIFICANCE AND IMPACT OF THE STUDY: A model describing the behaviour of fungal conidia is proposed.     

Effect of nitrogen levels of two cherry tomato cultivars on development,preference and honeydew production of Trialeurodes vaporariorum (Hemiptera: Aleyrodidae)

Two cherry tomato plant cultivars (Lycopersicon esculentum Miller, cultivars ‘Koko’ and ‘Pepe’) were supplied with high (395 ppm), medium (266 ppm) and low (199 ppm) concentrations of nitrogen to determine the influence of nitrogen fertilization on development, cultivar preference and honeydew production by greenhouse whiteflies, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae). The nitrogen, protein, and chlorophyll contents of tomato leaves were higher in the high nitrogen supplied plants than in the medium or low nitrogen supplied plants, but the sugar content showed an inverse relationship. The developmental times of eggs and nymphs decreased as the nitrogen concentrations increased in both cultivars. The preference of T. vaporariorum was compared by counting the number of eggs deposited on leaves in choice and non-choice tests. In the non-choice test, no significant nitrogen treatment effects were observed but the upper plant stratum was preferred for egg laying. In the choice test, there were significant main effects of cultivar and nitrogen concentration. T. vaporariorum laid eggs more on leaves of plants with higher nitrogen at the upper stratum. In both experiments, T. vaporariorum preferred the ‘Koko’ cultivar to the ‘Pepe’ cultivar. The honeydew production of T. vaporariorum nymphs increased with decreasing nitrogen treatment concentrations. The largest honeydew production was detected in the ‘Pepe’ cultivar grown at low nitrogen concentration. It is concluded that cultivar ‘Pepe’ had an advantage over ‘Koko’ in term of T. vaporariorum management program in tomato greenhouses.     

Effect of culture media, temperature and light on radial growth and pycnidium production of cowpea isolates of Phoma bakeriana

The effects of culture media, temperature and light on radial growth and pycnidium production of cowpea isolates (PB1, PB2, PB3) of Phoma bakeriana were investigated in vitro. There was no isolate effect on growth and pycnidium production but some evidence of a medium effect. Exceptionally high growth rate on two media corresponded with high pycnidium production; while low growth rates on other media were generally associated with high sporulation. There was a large temperature effect and an isolate effect on growth with a significant temperature by isolate interaction. Temperatures of 5°C, 10°C and 35°C inhibited pycnidium formation completely while the range 15–30°C had no effect on sporulation. Maximum growth and pycnidium production occurred at 25°C. Near-ultraviolet radiation seemed to have caused higher pycnidium counts than the other light sources.     

Effects of selected pesticides used against glasshouse tomato pests on colony growth and conidial germination of Paecilomyces fumosoroseus

Two isolates of a potential biological control agent, Paecilomyces fumosoroseus, for greenhouse whitefly, Trialeurodes vaporariorum, on tomato in greenhouses were tested for their compatibility with five fungicides (captan, dichlofluanid, iprodione, benomyl, and carbendazim) and six insecticides (malathion, carbaryl, primicarb, pymetrozine, cypermethrin, and deltamethrine) which are used on glasshouse tomato. Conidial germinations after 24 h and radial growths after 14 days of inoculation were determined in the presence of each pesticide at three concentrations (recommended rate, 0.1 and 10 times of the recommended rate by its manufacturer) and resultant variations were evaluated. Our results suggest that the tested pesticides, especially the fungicides, have potential to interfere with the success of P. fumosoroseus if they are to be employed together. The recommended concentration rate of primicarb and pymetrozine, and the lower concentrations of cypermethrin and deltamethrin were less hazardous than the others.     

Effect of temperature on sporulation of Neozygites floridana isolates from different climates and their virulence against the tomato red spider mite, Tetranychus evansi

The fungal pathogen Neozygites floridana Weiser and Muma has been evaluated as a classical biological candidate for introduction into Africa against the invasive tomato red spider mite Tetranychus evansi Baker and Pritchard. In this study, the effect of temperature on sporulation, germination and virulence of three isolates of N. floridana collected from T. evansi in three climatically distinct regions of Brazil and Argentina was determined. Six constant temperatures of 13 °C, 17 °C, 21 °C, 25 °C, 29 °C and 33 °C were tested for their effect on the ability of the three fungal isolates to sporulate, germinate and kill the mites. Six alternating-temperature regimes of 17-13 °C, 21-13 °C, 29-13 °C, 33-13 °C, 33-23 °C, 33-28 °C under a 12 h photophase were also tested to estimate virulence of the three isolates against T. evansi. The Vipos isolate discharged more conidia than isolates from Recife or Piracicaba at all temperatures and sporulation was strongly temperature dependent. Optimal sporulation rates were observed at 25 °C while optimal germination rates were observed at 25 °C and 29 °C. At 29 °C, the shortest mean survival time of T. evansi (3.16 days, 95% CI of 3.05-3.27) was observed for the isolate from Vipos, while the longest LT₅₀ (3.47 days, 95% CI 3.34-3.59) was observed for the isolate from Piracicaba. Mortality of mites increased as the differences between alternating day and night temperatures increased from 8 °C (21-13 °C), to 10 °C (33-23 °C), to 16 °C (29-13 °C), with smallest and highest temperature differences of 4 °C (17-13 °C) and 20 °C (33-13 °C), both producing low mortalities. The overall results suggest that the Vipos isolate is better adapted to a wider range of temperatures than the other isolates tested.     

Effects of exogenous nutrients on conidial germination and virulence against the silverleaf whitefly for two hyphomycetes

Exogenous protein and sugar sources were tested for their impact on conidial germination of two silverleaf whitefly (Bemisia argentifolii) pathogens: Beauveria bassiana and Paecilomyces fumosoroseus. In liquid culture, sugars stimulated only 5-27% germination of B. bassiana and < or =11% germination of P. fumosoroseus, whereas, yeast extract or peptone stimulated 95-100% germination. In the absence of additional nutrients, agar alone stimulated approximately 50% germination. Storing spores for different periods of time did not alter their general response to exogenous nutrients. When spores were germinated before being applied to third instar B. argentifolii, mortality was as much as 2.45 times greater and occurred more rapidly than that for fresh spores. For ungerminated conidia, the mean time to death from infection was 5.45 (SE = 0.16) and 4.74 (SE = 0.08) days for application rates of 37 and 144 conidia x mm(-2), respectively. When conidia were germinated before application, infection times dropped to 4.58 (SE = 0.16) and 4.45 (SE = 0.10) days, respectively. A likely explanation for the greater pathogenicity and virulence of germinated over ungerminated B. bassiana conidia is that only a fraction of the spores applied to whitefly nymphs actually germinate on the cuticle. For some specialized applications, such as greenhouse production systems, it may be beneficial to germinate spores immediately prior to application.     

Effect of temperature and water activity on spore germination and mycelial growth of three fungal biocontrol agents against water hyacinth (Eichhornia crassipes)

Aims: To determine the effect of water activity (a_w = 0·880–0·960) and temperature (15–35°C) on the percentage of viable conidia and mycelial growth of three biocontrol agents effective against water hyacinth in Mali: Alternaria sp. isolate Mlb684, Fusarium sacchari isolate Mln799 and Cadophora malorum isolate Mln715. Methods and Results: The fungi were grown in vitro on plates containing potato dextrose agar medium at different a_w values (glycerol being added to adjust the a_w). The percentage of viable conidia and radial growth rate decreased with decreasing water activity. Statistical analysis showed a significant effect of a_w, temperature and the a_w × temperature interaction on mycelial growth (P < 0·0001). Water activity emerged as the factor exerting the greatest influence. Differences were observed between the fungi tested, the C. malorum appearing more tolerant to low a_w and the F. sacchari more tolerant to high temperature (35°C). Growth models predicting the combined effect of a_w and temperature were developed and response surfaces generated, showing fairly good agreement with the experimental values. Conclusions: Our results confirm the previous finding that a_w has a greater influence than temperature on fungal growth. Under most conditions, variation of environmental factors has a detrimental influence on the percentage of viable conidia and mycelial growth rate of fungal isolates. Significance and Impact of the Study: The developed models may contribute to predicting the best environmental conditions for use of these fungi as effective biocontrol agents against water hyacinth.     

Temperature-dependent germination,growth and co-infection of Beauveria spp. isolates from different climatic regions

Biological control agents based on entomopathogenic fungi traditionally contain a single strain that is efficient under certain biotic and abiotic conditions. Since particularly abiotic conditions vary, biological control efficiency may become more resilient at extreme temperatures if two or more fungal strains are combined based on their adaptations to their original environment. Here we evaluated the in vitro temperature-dependent germination and growth rate for six Beauveria spp. isolates originating from either arctic or tropical regions. Isolates of arctic origin showed higher germination and growth rate at 8°C and 12°C than isolates from the tropics while the latter group showed highest germination and in vitro growth at 32°C. Three of the isolates belonging to Beauveria bassiana were further tested in vivo for temperature-dependent infection in the mealworm beetle Tenebrio molitor both individually and combined. The same amounts of conidia were used in all bioassays. Virulence was isolate dependent at all temperatures with no additional effect at the low (12°C) and high (32°C) temperatures of combinations of arctic and tropical isolates. The results therefore indicate that adaptations to abiotic conditions in the natural environment do not directly reflect the effect of biotic environment (such as host infection) under similar conditions. Selection of isolates for biocontrol agents should not be based solely on in vitro experiments, while isolate selection based on virulence should also include considerations of the abiotic conditions the isolates are expected to function.     

Application of Doehlert design to determine the combined effects of temperature, water activity and pH on conidial germination of Penicillium chrysogenum

AIMS: The influence of temperature, water activity and pH on the time necessary for germination of 90% of Penicillium chrysogenum conidia inoculated (T90) was determined. METHODS AND RESULTS: A new experimental device was developed for easy monitoring of the germination process. Experiments were carried out according to a Doehlert matrix at 11-31 degrees C, 0.86-0.98 water activity (a(w)) and pH 3.5-6.5. In these conditions, a second order polynomial relationship between T90 and the environmental factors was established for the different humectants used throughout this study (e.g. glycerol and sorbitol) with regression coefficients close to 0.97. CONCLUSIONS: For both humectants, the major effect of temperature and water activity on T90 was highlighted, whereas the effect of pH on T90 in these experimental conditions was not significant. The combined effect of temperature and water activity on T90 was also demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Both the experimental set-up and the Doehlert matrix were well suited to determine the influence of environmental factors on mould germination.     

Effect of temperature,hydrogen ion concentration and osmotic potential on oospore germination of fivePythium spp. isolated from pond water

Oospore germination occurred over a temperature ranging of 15–35°C forPythium coloratum, 10–35°C forP. diclinum, 15–30°C forP. dissotocum, 7–30°C forP. monospermum, and 10–30°C forP. pleroticum. Optimum temperature was 25°C for all species tested. In case of pH, oospore germination occurred over a range of 4.76–8.55 with an optimum of 6.40–7.40. The least germination occurred at pH 4.76 forP. coloratum, P. diclinum, P. monospermum andP. pleroticum, whileP. dissotocum germinated from pH 5.02. Oospores of the all tested pythia were able to germinate at –0.13 to –1.65 MPa and could not germinate at –3.40 MPa, with the highest germination rate at –0.27 to –0.47 MPa. The effect of temperature, pH and osmotic potential on oospore germination was discussed in relation to pollution of pond water.     

Effect of temperature and water activity on growth and ochratoxin A production boundaries of two Aspergillus carbonarius isolates on a simulated grape juice medium

Aims: To develop and validate a logistic regression model to predict the growth and ochratoxin A (OTA) production boundaries of two Aspergillus carbonarius isolates on a synthetic grape juice medium as a function of temperature and water activity (a_w). Methods and Results: A full factorial design was followed between the factors considered. The a_w levels assayed were 0·850, 0·880, 0·900, 0·920, 0·940, 0·960, 0·980 and the incubation temperatures were 10, 15, 20, 25, 30, 35 and 40°C. Growth and OTA production responses were evaluated for a period of 25 days. Regarding growth boundaries, the degree of agreement between predictions and observations was >99% concordant for both isolates. The erroneously predicted growth cases were 3·4–4·1% false‐positives and 0·7–1·4% false‐negatives. No growth was observed at 10°C and 40°C for all a_w levels assayed, with the exception of 0·980 a_w/40°C, where weak growth was observed. Similarly, OTA production was correctly predicted with a concordance rate >98% for the two isolates with 0·7–1·4% accounting for false‐positives and 2·0–2·7% false‐negatives. No OTA production was detected at 10°C or 40°C regardless of a_w, and at 0·850 a_w at all incubation temperatures. With respect to time, the OTA production boundary shifted to lower temperatures (15–20°C) as opposed to the growth boundary that shifted to higher temperature levels (25–30°C). Using two literature datasets for growth and OTA production of A. carbonarius on the same growth medium, the logistic model gave one false‐positive and three false‐negative predictions out of 68 growth cases and 13 false‐positive predictions out of 45 OTA production cases. Conclusions: The results of this study suggest that the logistic regression model can be successfully used to predict growth and OTA production interfaces for A. carbonarius in relation to temperature and a_w. Significance and Impact of the Study: The proposed modelling approach helps the understanding of fungal‐food ecosystem relations and it could be employed in risk analysis implementation plans to predict the risk of contamination of grapes and grape products by A. carbonarius.     

The cAMP-dependent signaling pathway and its role in conidial germination, growth, and virulence of the gray mold Botrytis cinerea

In Botrytis cinerea, some components of the cAMP-dependent pathway, such as alpha subunits of heterotrimeric G proteins and the adenylate cyclase BAC, have been characterized and their impact on growth, conidiation, germination, and virulence has been demonstrated. Here, we describe the functions of more components of the cAMP cascade: the catalytic subunits BcPKA1 and BcPKA2 and the regulatory subunit BcPKAR of the cAMP-dependent protein kinase (PKA). Although Deltabcpka2 mutants showed no obvious phenotypes, growth and virulence were severely affected by deletion of both bcpka1 and bcpkaR. Similar to Deltabac, lesion development of Deltabcpka1 and DeltabcpkaR was slower than in controls and soft rot of leaves never occurred. In contrast to Deltabac, Deltabcpka1 and DeltabcpkaR mutants sporulated in planta, and growth rate, conidiation, and conidial germination were not impaired, indicating PKA-independent functions of cAMP. Unexpectedly, Deltabcpka1 and DeltabcpkaR showed identical phenotypes, suggesting the total loss of PKA activity in both mutants. The deletion of bcras2 encoding the fungal-specific Ras GTPase resulted in significantly delayed germination and decreased growth rates. Both effects could be partially restored by exogenous cAMP, suggesting that BcRAS2 activates the adenylate cyclase in addition to the Galpha subunits BCG1 and BCG3, thus influencing cAMP-dependent signal transduction.     

Water activity and temperature effects on germination and growth of Eurotium amstelodami, E. chevalieri and E. herbariorum isolates from bakery products

This study investigated the effect of temperature (5-30 degrees C), water activity (0.775-0.90 aw) and their interactions on the temporal rates of germination and mycelial growth of three species of Eurotium on flour wheat sucrose medium. Germination was quite rapid at aw >0.85, with an almost linear increase with time for all isolates. However, under more extreme water stress, germination was slower. The aw minima for germination were usually lower than those for growth and varied with temperature. The effect of aw x temperature interactions on the lag phases (h) prior to germination and on the germination rates (h-1) were predicted using the Gompertz model modified by Zwietering. Eurotium spp. had shown short lag times at 0.90 aw over a wide range of temperatures. At marginal temperatures, lag phases were significantly longer, especially at >15 degrees C. The temperature x aw profiles for mycelial growth varied between species in terms of rates (mm d(-1)). Predictions of the effect of important environmental factors, such as temperature, aw and their interactions on lag times to germination, germination rates and mycelial growth, are important in the development of hurdle technology approaches to predict fungal spoilage in food products.     

Effects of successive subculturing on stability, virulence, conidial yield, germination and shelf-life of entomopathogenic fungi

Aims: To determine the stability and conidial yield of two strains of the entomopathogenic fungus Metarhizium anisopliae and one strain of M. brunneum, being developed for the control of insect pests. Methods and Results: The conidial yields and the shelf‐life of the conidia of two commercially viable strains of M. anisopliae V275 (=F52) and ARSEF 4556 and one strain of M. brunneum (ARSEF 3297) were determined after harvesting conidia from in vitro subcultures on Sabouraud dextrose agar (SDA) and broken basmati rice. The strains were stable and showed no decline in virulence against Tenebrio molitor, even when subcultured successively 12 times on SDA. Conidia‐bound Pr1 protease activity decreased in conidia harvested from SDA and mycosed cadavers after the 1st subculture, but increased in conidia produced on rice. The C:N ratio of conidia from mycosed cadavers was lower than that of conidia from rice or SDA. Irrespective of the number of subcultures, strain ARSEF 4556 produced significantly higher conidial yields than ARSEF 3297 and V275. The 12th subculture of V275 and ARSEF 3297 produced the lowest conidial yield. Shelf‐life studies showed that conidia of strain ARSEF 4556 had a higher conidial viability than V275 and ARSEF 3297 after 4 months, stored at 4°C. Conclusions: The current study shows that determining strain stability and conidial yield through successive subculturing is an essential component for selecting the best strain for commercial purposes. Significance and Impact of the Study: This is the first study to compare quality control parameters in the production of conidia on rice, and it shows that the level of Pr1 is comparatively high for inoculum produced on rice.     

Effect of Lactobacillus plantarum on germination and growth of tomato seedlings

Significant stimulation of germination of tomatoes (Lycopersicon esculentum Mill. cv Odessa plum) with poor initial germination capacity was achieved by soaking their seeds during 6 h in suspensions of nine out of ten Lactobacillus plantarum strains tested. The increase in percentage of germination ranged from 15.1 to 7.6 %. Lengths of shoots, main and lateral roots and development of root hairs increased after inoculation of seeds with studied L. plantarum strains. The treatments of seeds with L. plantarum expanded shoot lengths by 16.4–18.2 %. Stimulation of main root lengths in the tested tomato seedlings varied from 10.5 to 31.4 %, and the stimulation of lateral roots from 28.6 to 32.2 %. Lactobacilli from different strains exert different effects on the seedlings: for instance, inoculation with certain strains resulted in the stimulation of the main root growth and the inoculation with another strain stimulated shoot growth or growth of the lateral roots. It is the first report about plant-stimulating activities of L. plantarum from different ecological niches (plant surfaces, surfaces of edible mushrooms, dairy products) in the absence of nutritional compounds.