Microencapsulating aerial conidia of Trichoderma harzianum through spray drying at elevated temperatures

Conidia of Trichoderma harzianum produced from either solid or liquid fermentation must be dried to prevent spoilage by microbial contamination, and to induce dormancy for formulation development and prolonged self-life. Drying conidia of Trichoderma spp. in large scale production remains the major constraint because conidia lose viability during the drying process at elevated temperatures. Moreover, caking must be avoided during drying because heat generated by milling conidial chunks will kill conidia. It is ideal to dry conidia into a flow-able powder for further formulation development. A method was developed for microencapsulation of Trichoderma conidia with sugar through spray drying. Microencapsulation with sugars, such as sucrose, molasses or glycerol, significantly (P < 0.05) increased the survival percentages of conidia after drying. Microencapsulation of conidia with 2% sucrose solution resulted in the highest survival percentage when compared with other sucrose concentrations and had about 7.5 × 10¹⁰ cfu in each gram of dried conidia, and 3.4 mg of sucrose added to each gram of dried conidia. The optimal inlet/outlet temperature setting was 60/31 °C for spray drying and microencapsulation. The particle size of microencapsulated conidia balls ranged from 10 to 25 μm. The spray dried biomass of T. harzianum was a flow-able powder with over 99% conidia, which could be used in a variety of formulation developments from seed coatings to sprayable formulations.     

Culture medium improvement for Isaria fumosorosea submerged conidia production

Submerged conidia and blastospores of the entomopathogenic fungus Isaria fumosorosea are produced in several liquid culture media. However, yields and the ecological fitness of these propagules vary according to culture media composition. In most culture media, hyphae, blastospores and submerged conidia are white but we found that in some media they develop a brown pigmentation. A dark pigment was extracted from brown-pigmented propagules and analyzed by IR spectroscopy. Adsorption bands coincided to those characteristics of melanins.Hadamard's matrices were employed in order to increase submerged conidia yields and brown pigmentation of fungal propagules. Media containing 20–30 mg/l of FeSO₄·7H₂O and 6–12 mg/l of CuSO₄·5H₂O allowed reaching the highest pigmentation (9 in a hedonic scale). A maximal concentration of submerged conidia of 1.0 (±1.2) × 10¹² cell/l was achieved after 120 h of liquid culture in a improved culture medium, containing 25 ml/l of Polyethylene glycol (MW 200), substance which enhanced submerged conidia production, reducing free mycelia or mycelial pellets formation. In the improved medium, it was estimated that more than 60% of produced biomass corresponded to submerged conidia and blastospores, while in other media, mycelia were the main product (80–97%).     

Laboratory and semi-field evaluation of Beauveria bassiana (Ascomycota: Hypocreales) against the lettuce aphid,Nasonovia ribisnigri (Hemiptera: Aphididae)

The lettuce aphid, Nasonovia ribisnigri (Mosley), is an economically important pest of lettuce worldwide. The entomopathogenic fungus Beauveria bassiana strain GHA has recently been reported as a potential biocontrol candidate for use against the lettuce aphid. This study provides information on the mortality inflicted by B. bassiana when applied against different life stages of the lettuce aphid under laboratory conditions and how fungus infection affects the aphid fecundity. In addition, temporal changes in persistence of fungus inoculum applied to foliage of young lettuce plants under semi-field conditions was analysed. Immature life stages were generally the least susceptible to fungal infection and the susceptibility of all stages was dose-dependent, with the highest mortality occurring at the highest dose. B. bassiana significantly affected the rate of nymph production by the lettuce aphid, with the highest effect seen when the alatoid fourth instar of N. ribisnigri was inoculated with B. bassiana. The persistence of B. bassiana conidia on lettuce foliage was not influenced by leaf position. Within 5 days, the cumulative percentage decline in the conidial population was 38% which declined further to 92% and 99% on day 11 and 20 post-spraying, respectively. In accordance, the infectivity to second instar lettuce aphid nymphs of B. bassiana conidia deposited on leaves declined according to an exponential decay model predicting an intercept of 0.59 ± 0.03 (S.E), a reduction in aphid mortality at a rate of 11% with each increasing day after fungal application and a fungus half-life of 6.34 ± 0.69 days.     

Effects of fluctuating moisture and temperature regimes on the persistence of quiescent conidia of Isaria fumosorosea

Conidia of Isaria fumosorosea were submitted to three regimes of temperature and moisture to simulate microclimatic conditions which prevail in temperate (43% RH and 28 °C to 98% RH and 15 °C), subtropical (75% RH and 35 °C to 98% RH and 25 °C), and arid areas (13% RH and 40 °C to 33% RH and 15 °C) with daily fluctuating cycles. Germination, conidial viability, and virulence to Spodoptera frugiperda larvae were less affected after 20 days exposure under temperate cycling conditions than under arid and subtropical conditions. Exposure of conidia for 20 days to constant nocturnal simulated conditions of any tested regime weakly affected conidial persistence, whereas diurnal conditions exerted the most detrimental effects of high temperatures. However, when tested at both 45 °C and 50 °C at 33% RH for 160 h, the persistence of I. fumosorosea conidia was relatively higher than expected. These results emphasize that climatic conditions prevailing in environments and ecological fitness of fungal isolates have to be taken into account for assessing microbial control strategies.     

Behavioral and electrophysiological responses of Coptotermes formosanus Shiraki towards entomopathogenic fungal volatiles

Termites adjust their response to entomopathogenic fungi according to the profile of fungal volatile organic compounds (VOCs). This study demonstrates the pathogenicity of Metarhizium anisopliae, Beauveria bassiana and Isaria fumosorosea (=Paecilomyces fumosoroseus) towards the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). Using no-choice assays, M. anisopliae was found to be highly virulent (LT₅₀ 3.10 d) when compared to B. bassiana (LT₅₀ 6.62 d) and I. fumosorosea (LT₅₀ 12.39 d). Also using choice assays, the foraging behavior of C. formosanus was determined in the presence of pathogenic fungi. The highly pathogenic fungi (M. anisopliae) elicited a repellent response, causing most of the termites to forage in a safe zone farthest from the fungal source. This repellency resulted in relatively low mortality similar to the controls. The repellency of M. anisopliae conidia can be used to protect human belongings and timber from termites. While I. fumosorosea cultures were not repellent to C. formosanus workers, the termites were highly susceptible to infection. Electroantennographic responses of workers showed approximately 47% and 78% lower level of response to conidia of B. bassiana and I. fumosorosea, respectively, as compared to M. anisopliae. The VOC profile of repellent cultures of M. anisopliae mainly consisted of paraffins (60.97%), while the major proportion of the I. fumosorosea profile consisted of branched and cyclic alkanes (84.41%). From the above findings, we conclude that the incorporation of I. fumosorosea may increase the control potential of bait.     

Photosynthetic pigments,morphology and leaf gas exchange during ex vitro acclimatization of micropropagated CAM Doritaenopsis plantlets under relative humidity and air temperature

The effect of relative humidity (RH) and temperature on CO₂ assimilation (An), stomatal conductance (Sc), transpiration rate (Tr), chlorophyll content, fresh and dry weight, leaf length, leaf area, leaf width, formation of new root and survival rate have been assayed in Doritaenopsis in growth chamber after 1 month of acclimatization. Reduced growth was observed at below and above 25 °C whereas it was increased with increasing humidity. Relative water content (RWC) was decreased at 50% and 70% humidity after second day of transfer and recovered completely with the progression of acclimatization. RWC also reduced at high temperature but recovered slowly and a gradual decrease of RWC was observed at 15 °C. A visual symptom of severe leaf tip burn was observed at 50–70% humidity and at 35 °C during acclimatization. At 15 °C and 50% humidity sudden decrease of photosynthetic efficiency (F_v/F_m) was observed, which could not recover in temperature treated plantlets during acclimatization period. Chlorophyll content increased with increasing humidity and at 15 and 35 °C chlorophyll content was decreased compared to 25 °C. Chlorophyll a/b ratio was unchanged while total chlorophyll/carotenoids ratio was increased from low to high temperature. Exposure of plantlets to high temperature led to a noticeable decrease in An, Sc and Tr, and at 15 °C they were more decreased whereas significant differences were not observed in the parameters tested under humidity after 25 days of acclimatization. During daytime at 15 °C, increase in An, Sc and Tr indicates the plantlets adaptability in the new environment. The peroxidase activity remained unaffected in all humidity stress whereas low temperature increased the peroxidase activity compared to high temperature. These finding suggests that photosynthetic properties was greatly affected by air temperature conditions with a reduction of An, Sc and Tr at 15 and 35 °C compared to humidity stress that played a greater role in limiting photosynthesis.     

The importance of relative humidity and trophic resources in governing ecological niche of the invasive carabid beetle Merizodus soledadinus in the Kerguelen archipelago

Comprehensive studies to identify species-specific drivers of survival to environmental stress, reproduction, growth, and recruitment are vital to gaining a better understanding of the main ecological factors shaping species habitat distribution and dispersal routes. The present study performed a field-based assessment of habitat distribution in the invasive carabid beetle Merizodus soledadinus for the Kerguelen archipelago. The results emphasised humid habitats as a key element of the insect’s realised niche. In addition, insects faced food and water stress during dispersal events. We evaluated quantitatively how water availability and trophic resources governed the spatial distribution of this invasive predatory insect at Îles Kerguelen. Food and water stress survival durations [in 100%, 70%, and 30% relative humidity (RH) conditions] and changes in a set of primary metabolic compounds (metabolomics) were determined. Adult M. soledadinus supplied with water ad libitum were highly tolerant to prolonged starvation (LT₅₀ = 51.7 ± 6.2 d). However, food-deprived insect survival decreased rapidly in moderate (70% RH, LT₅₀ = 30.37 ± 1.39 h) and low (30% RH, LT₅₀ = 13.03 ± 0.48 h) RH conditions. Consistently, body water content decreased rapidly in insects exposed to 70% and 30% RH. Metabolic variation evidenced the effects of food deprivation in control insects (exposed to 100% RH), which exhibited a progressive decline of most glycolytic sugars and tricarboxylic acid cycle intermediates. Most metabolite levels were elevated levels during the first few hours of exposure to 30% and 70% RH. Augmented alanine and lactate levels suggested a shift to anaerobic metabolism. Simultaneously, peaks in threonine and glycolytic sugars pointed to metabolic disruption and a progressive physiological breakdown in dehydrating individuals. Overall, the results of our study indicate that the geographic distribution of M. soledadinus populations is highly dependent on habitat RH and water accessibility.     

Two new species of nematode-trapping fungi: relationships inferred from morphology,rDNA and protein gene sequence analyses

Two new nematode-trapping fungi, Dactylellina sichuanensis and D. varietas from China, which capture nematodes by both adhesive knobs and non-constricting rings, are described and illustrated. D. sichuanensis is characterized by both adhesive knobs and non-constricting rings, solitary conidiophores and 3-(4)-6 septate conidia, as compared with species such as D. appendiculata, D. candida, D. leptospora and D. lysipaga. Although D. sichuanensis shares similar types of trapping devices, the presence of simple conidiophores and spindle-shaped conidia with these species, it can be distinguished by its larger conidia and presence of more than four septa. In D. sichuanensis, a single conidium is born at the tip of conidiophore, while in D. candida, 3–10 conidia are born near the apex of conidiophore in a capitate arrangement. It produces spindle-shaped conidia with 3–6 septa, whereas D. leptospora produces cylindrical-shaped conidia with 5–15 septa. D. appendiculata captures nematodes by adhesive knobs whereas D. sichuanensis captures nematodes by both adhesive knobs and non-constricting rings. They also differ in conidial size (35–82.5 μm in D. sichuanensis as compared with 57–108 μm in D. appendiculata). D. varietas is characterized by conidiophores that are branched at right-angles, and elongate to fusoid conidia, with 7–8 septa (more than 25% of which are curved). D. varietas resembles D. asthenopaga, Dactylella oxyspora and Monacrosporium multiseptatum, but has elongate-fusoid conidia, whereas D. asthenopaga possesses obconical or clavate conidia. D. varietas forms both adhesive knobs and non-constricting rings whereas Dactylella oxyspora does not produce any trapping device. M. multiseptatum differs from D. varietas in having larger conidia with an inflated middle cell. Phylogenetic analyses based on nuclear and protein coding DNA sequences (18 S, and a combined 28 S + 5.8 S + β-tubulin dataset) indicate that these two taxa should be assigned to the family Orbiliaceae.     

Assessment of oral virulence against Spodoptera litura,acquired by a previously non-pathogenic Metarhizium anisopliae isolate,following integration of a midgut-specific insecticidal toxin

All entomopathogenic fungi infect insects by direct penetration through the cuticle rather than per os through the gut. Genetic transformation can confer fungi with per os virulence. However, unless the recipient isolate is nonpathogenic to the target insect, mortality caused by a transgenic isolate cannot be attributed solely to oral virulence due to the potential for some simultaneous cuticular infection. Here, a Metarhizium anisopliae wild-type isolate (MaWT) nonpathogenic to Spodoptera litura was genetically engineered to provide a transformed isolate (MaVipT31) expressing the insect midgut-specific toxin Vip3Aa1. Toxin expression was confirmed in MaVipT31 hyphae and conidia using Western blotting. Mortality, leaf consumption and body weight of S. litura larvae (instars I–IV) exposed to a range of concentrations of MaWT conidia were not significantly different to controls although the number of conidia ingested by surviving larvae during the bioassay ranged from 2.3 × 10⁵ (instar I) to 8.1 × 10⁶ (instar IV). In contrast, consumption of MaVipT31 conidia caused high mortalities, reduced leaf consumption rates and decreased body weights in all instars evaluated, demonstrating that oral virulence had been acquired by MaVipT31. Larval mortalities were much more dependent on the number of MaVipT31 conidia ingested than the duration of time spent feeding on conidia-treated leaves (r²: 0.83–0.94 for instars I–IV). LC₅₀ and LT₅₀ trends for MaVipT31 estimated by time-concentration-mortality modeling analyses differed greatly amongst the instars. For 50% kill to be achieved, instar I larvae required 3, 4 and 5 days feeding on the leaves bearing 103, 28 and 8 conidia/mm² respectively; instar IV larvae required 6, 7 and 8 days feeding on leaves bearing 1760, 730 and 410 conidia/mm² respectively. Our results provide a deeper insight into the high oral virulence acquired by an engineered isolate and highlight its great potential for biological control.     

Alginate embedding and subsequent sporulation of in vitro-produced Conidiobolus thromboides hyphae using a pressurised air-extrusion method

Conidiobolus thromboides is an entomophthoralean fungus with potential as a biological control agent of aphids. However, its application in biological control is limited due to its formulation requirements. The objective of this study was to develop and optimise a novel air-extrusion method to embed C. thromboides hyphae at high density in alginate pellets. An orthogonal experimental design was used to investigate selected combinations of parameters known to affect hyphal density within pellets. The diameter of pellets produced, and the calculated density of hyphae within them, ranged from 0.18 ± 0.09 to 3.17 ± 0.06 mm and from 0.02 to 350.56 mg/mm³ respectively. These data were used to predict the optimal parameter combination to deliver the greatest density of hyphae of C. thromboides per pellet: 1% sodium alginate, a 1:2 ratio of hyphae to sodium alginate, an orifice diameter of 0.232 mm and an air pressure of 0.05 MPa. Pellets made under the optimal conditions predicted produced a mean total of 4.3 ± 0.6 × 10⁵ conidia per pellet at 100% relative humidity which was significantly greater than the mean total number of conidia produced from infected aphid cadavers of comparable size (9.35 ± 0.85 × 10⁴) (p < 0.001). In conclusion, air-extrusion embedding appears to be a promising method for formulating in vitro-produced hyphae of C. thromboides for use in biological control.     

Constraints in the identification and interpretation of ozone as a significant predictor of effects on the supersensitive indicator Nicotiana tabacum Bel-W3 in biomonitoring studies

Biomonitoring by means of the supersensitive cultivar Nicotiana tabacum Bel-W3 is frequently used to obtain information on ozone effects on plants and estimates of ozone exposure. However, most of biomonitoring surveys do not account for other environmental variables (predictors in a statistical model) and their inherent multicollinearity with ozone. We tested the relative role of different predictors (fixed: time and site; random: ozone, temperature and humidity) on height growth and on the development of visible foliar symptoms of N. tabacum Bel-W3 plants. To do this, we investigated a relatively small area (256 km²), used a random design at every stage of the survey, controlled watering and protected plants from direct solar radiation and wind. QA/QC procedures were adopted at every stage of the investigation. Linear correlation shows that Leaf Injury Index (LII) and height increment (H.I.) positively related to ozone concentration, elevation and temperature, and negatively to relative humidity. All the predictors correlate to each other. However, relationships between response and ozone vary with the site and the monitoring week. The effect of the random factor “ozone” in combination with fixed factors “site” and “time” on the response variables was therefore formally investigated using the ANCOVA model. Besides ozone, the interactions “ozone × site” and “ozone × time” resulted always significant (0.001 < P < 0.05). While the factor “time” emphasize the inherent development of injury and growth through time, the interaction “ozone × site” pointed out the importance of local conditions. When watering, solar radiation, wind and plant characteristics were controlled, the remaining site-specific covariates of interest were temperature (T) and humidity (RH). When T and RH were accounted for by means of partial correlation analysis, no significant relationship was found between ozone and LII. On the other side, O₃ and RH resulted significant for both absolute and relative height increment. In short, LII seemed to be not solely dependent on ozone, T and RH, but showed to integrate their combined effect. On the other side, H.I. seemed to be favoured by high RH and T, and depressed by high ozone. Based on the above results, we recommend caution when handling bioindicator data: if the purpose is to infer ozone concentrations by leaf injury data, results may be affected by a serious bias, as the frequently reported correlations may be partly an artefact due to co-variation between predictors.     

Cellular damage during drying and storage of Trichoderma harzianum spores

Factors that cause cellular damage during the drying and storage of Trichoderma harzianum conidia were independently studied to determine their effects on spore viability. Specifically, thermal stress and dehydration levels (water activity, a_w = 0.1–0.7) were assessed for their effect on spore survival. In addition, environmental conditions, such as water activity and temperature, were evaluated during storage of the spores. T. harzianum spores produced in liquid culture are highly sensitive to thermal stress, but dehydration does not seem to be a factor that influences spore death during desiccation. An inverse correlation between spore survival and the specific concentration of malondialdehyde (MDA) was observed during storage, especially when the conidia moisture levels were lower than the monolayer moisture levels. We prepared spore suspensions without additives and spray-dried the samples. Our data showed that reduced sample viability was mainly caused by the temperature of the drying process, an effect that appears to be independent of water activity.     

Efficacy of the antagonist Aureobasidium pullulans PL5 against postharvest pathogens of peach,apple and plum and its modes of action

The efficacy of Aureobasidium pullulans PL5 against different postharvest pathogens of fruits (Monilinia laxa on plums and peaches, Botrytis cinerea and Penicillium expansum on apples) were evaluated under storage conditions when applied at 10⁸ cells ml⁻¹ and their interactions were studied in vitro and in vivo to discover the possible modes of action. Under 1.2 °C and 95% relative humidity (RH) for 28 days, the efficacy of PL5 against M. laxa on plums was 45%, reducing disease incidence from 78% to 43%. Under 1 °C and 95% RH for 21 days, the efficacy against M. laxa on peaches was 63%, reducing disease incidence from 79% to 29%. Under 4 °C and 95% RH for 45 days, the efficacy against B. cinerea and P. expansum on apples was 56% and 46%, respectively. In Lilly–Barnett minimal salt medium with the fungal cell walls of pathogens as sole carbon source, PL5 produced β-1,3-glucanase, exo-chitinase and endo-chitinase. Nutrient concentrations had significant effect on pathogen growth reduction by PL5. No attachment was observed in antagonist–pathogen interactions in vitro or in vivo. PL5 completely inhibited pathogen spore germination in PDB at 10⁸ cells ml⁻¹, whereas at 10⁶ cells ml⁻¹ the efficacy was significantly decreased. However, inactivated cells and culture filtrate of PL5 had no effect on pathogen spore germination and germ tube elongation. Our results showed that A. pullulans PL5 could be introduced in commercial formulations to control postharvest pathogens on fruits and its activity was based on secretion of lytic enzymes and competition for nutrients.     

Stature,body mass,and brain size: A two-million-year odyssey

Physical size has been critical in the evolutionary success of the genus Homo over the past 2.4 million-years. An acceleration in the expansion of savannah grasslands in Africa from 1.6 Ma to 1.2 Ma witnessed concomitant increases in physical stature (150–170 cm), weight (50–70 kg), and brain size (750–900 cm³). With the onset of 100,000 year Middle Pleistocene glacial cycles (“ice ages”) some 780,000 years ago, large-bodied Homo groups had reached modern size and had successfully dispersed from equatorial Africa, Central, and Southeast Asia to high-latitude localities in Atlantic Europe and North East Asia. While there is support for incursions of multiple Homo lineages to West Asia and Continental Europe at this time, data does not favour a persistence of Homo erectus beyond ～400,000 years ago in Africa, west and Central Asia, and Europe. Novel Middle Pleistocene Homo forms (780,000–400,000 years) may not have been substantially taller (150–170 cm) than earlier Homo (1.6 Ma–800,000 years), yet brain size exceeded 1000 cm³ and body mass approached 80 kg in some males. Later Pleistocene Homo (400,000–138,000 years) were ‘massive’ in their height (160–190 cm) and mass (70–90 kg) and consistently exceed recent humans. Relative brain size exceeds earlier Homo, yet is substantially lower than in final glacial H. sapiens and Homo neanderthalensis. A final leap in absolute and relative brain size in Homo (300,000–138,000 years) occurred independent of any observed increase in body mass and implies a different selective mediator to that operating on brain size increases observed in earlier Homo.     

Influence of the operating conditions on lipase-catalysed synthesis of ricinoleic acid estolides in solvent-free systems

In this work, the synthesis of ricinoleic acid estolides, also named polyricinoleic acid (PR), in two different solvent-free reaction systems, catalysed by immobilised Candida rugosa lipase is described. First, the esterification was performed in an open air jacketed batch reactor and the influence of amount of enzyme, temperature and initial water content was tested. The best results were obtained when 5 g of soaked immobilised derivative was used as biocatalyst, and the reaction was carried out at 40 °C. It was observed that environmental relative humidity plays an important role in the enzymatic synthesis of ricinoleic acid estolides and, given that this parameter takes on a wide range of values depending on the season, it is considered the main cause of the poor reproducibility achieved in the open air reactor. To solve this problem, the ricinoleic acid estolides were synthesised under controlled atmosphere in a vacuum reactor with dry air intake. The optimum drying time of 7 h was selected. In these conditions, PR with an acid value of 57.5 mg KOH/g was synthesised in 48 h of reaction and the results were entirely reproducible.     

Biological control of Botrytis bunch rot in Atlantic climate vineyards with Candida sake CPA-1 and its survival under limiting conditions of temperature and humidity

Candida sake CPA-1 is an antagonistic yeast that has previously been shown to effectively control Botrytis bunch rot in grapes. The efficacy of biological control agents is dependent on their survival, which may also depend on climatic conditions. However, few studies have evaluated the effect of abiotic factors affecting the survival of biological control agents, such as temperature (T) or relative humidity (RH). In this study, efficacy of C. sake (5 × 10⁷ CFU mL⁻¹), which was applied with the additive Fungicover (FC; 50 g L⁻¹), was tested against BBR in the laboratory and in field trials under the Atlantic climate conditions of the Bordeaux region (France). The study also evaluated the survival of C. sake under T and RH regimes simulated in climatic chambers. Two or five applications of C. sake plus FC during the growing season significantly reduced BBR severity at harvest by 48% and 82%, respectively, when compared to the control. Similar reductions were achieved after inoculation with selected virulent Botrytis cinerea strains (75% compared to control) in laboratory experiments. C. sake populations showed minimal decreases between field applications and were favored by simulated Atlantic climate conditions. The survival pattern of C. sake exposed to 40 and 45 °C combined with 30% and 100% of RH was described, demonstrating a sharp decrease during the first 24 h. Allowing 48 h for C. sake to incubate and become established on fruits prior to the exposure to 40 °C and 30% RH increased survival (P < 0.05). These results confirm the efficacy of treatment with C. sake plus FC under favorable climatic conditions for BBR development, while survival studies may help to improve the survival and efficacy of yeast BCAs, such as C. sake CPA-1.     

Quality assessment of Riptortus pedestris (Hemiptera: Alydidae) eggs cold-stored at different temperature and relative humidity regime

Supplementation of host resource can be more economical method for the biological control of insect pest compared to direct release of adult parasitoids. Periodical release of non-viable cold-stored eggs of Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) has been found to enhance parasitism of this pest in soybean fields. To find the optimum environmental conditions for cold storage of these host eggs, we evaluated nine different combinations of temperature (2, 6, and 10 °C) and relative humidity (high 90–95%, medium 70–75%, and low 30–35%). After 30 d of cold-storage, eggs were weighed and held at 26.6 °C and 75% relative humidity for 8 d before testing. To test the eggs’ suitability as hosts following cold storage, females of Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) were released individually onto batches of eggs, and parasitization rates and the development, emergence, sex ratio, adult longevity, and size of parasitoid progeny were examined. Eggs stored at high relative humidity showed less weight loss than those stored at low relative humidity. The number of eggs parasitized was highest (5.9/15) on eggs stored at 6 °C and high relative humidity. Developmental times and adult emergence were optimal on host eggs stored at 2 °C and high relative humidity. A significantly lower proportion of eggs produced male parasitoids when eggs were stored at 2 or 6 °C. Adult longevity was not affected by egg storage conditions, but adult size of progeny decreased in eggs stored at 10 °C. In conclusion, eggs of R. pedestris stored below 6 °C and with a high relative humidity maintained the best quality for parasitization by O. nezarae.     

Reproducibility of a non-invasive ultrasonic technique of tendon force measurement,determined in vitro in equine superficial digital flexor tendons

A non-invasive ultrasonic (US) technique of tendon force measurement has been recently developed. It is based on the relationship demonstrated between the speed of sound (SOS) in a tendon and the traction force applied to it. The objectives of the present study were to evaluate the variability of this non-linear relationship among 7 equine superficial digital flexor (SDF) tendons, and the reproducibility of SOS measurements in these tendons over successive loading cycles and tests. Seven SDF tendons were equipped with an US probe (1 MHz), secured in contact with the skin overlying the tendon metacarpal part. The tendons were submitted to a traction test consisting in 5 cycles of loading/unloading between 50 and 4050 N. Four tendons out of the 7 were submitted to 5 additional cycles up to 5550 N. The SOS-tendon force relationships appeared similar in shape, although large differences in SOS levels were observed among the tendons. Reproducibility between cycles was evaluated from the root mean square of the standard deviations (RMS-SD) of SOS values observed every 100 N, and of force values every 2 m/s. Reproducibility of SOS measurements revealed high between successive cycles: above 500 N the RMS-SD was less than 2% of the corresponding traction force. Reproducibility between tests was lower, partly due to the experimental set-up; above 500 N the difference between the two tests stayed nevertheless below 15% of the corresponding mean traction force. The reproducibility of the US technique here demonstrated in vitro has now to be confirmed in vivo.     

Efficacy assessment of Pichia guilliermondii strain Z1, a new biocontrol agent,against citrus blue mould in Morocco under the influence of temperature and relative humidity

The interactions of Penicillium italicum, which causes blue mould, and antagonistic yeast Pichia guilliermondii strain Z1 were examined in controlled environments, to determine the influence of relative humidity (RH) (45%, 75%, 85%, 98%, and 100%) and temperature (T) (5, 10, 15, 20, and 25 °C). All main effects and interactions were significant (P ? 0.05), with the exception of interactions RH×T and strain Z1 (BCA)×RH×T. In the pathogen control, the lesion diameter of blue mould developed under all environmental conditions but was the largest at a RH range between 98% and 100%, independent of the temperature. The efficacy of strain Z1 appeared to be independent of the environment and reduced disease incidence by more than 85% in all environmental conditions. Rapid colonization of the antagonistic yeast strain Z1 on citrus wounded sites was recorded during the first week at 5 °C. Colonization then stabilized at ±6.9 × 10⁶ CFU/ml for 30 days. This indicates that P. guilliermondii is able to adapt itself and colonize the wound sites prior to the arrival of the pathogen, displaying greater efficiency than when colonizing wounds after pathogen. The antagonist was capable of growing in low concentrations of orange juice (0.1–5%), with greatest growth at 5%. Applying strain Z1 (1 × 10⁸ CFU/ml) as a formulated product significantly reduced the incidence of infected fruits and the percentage of infected wounds relative to the pathogen control. Disease control with formulated product (45%) was slightly lower than that obtained with thiabendazole (20%) or strain Z1 culturable cells (25%). These results suggest that strain Z1 may be a useful BCA for control of blue mould under varying environmental conditions, and control may be enhanced by combining with other eco-friendly post-harvest treatments or improved formulation.