In vitro water stress bioassays with the nematophagous fungus Pochonia chlamydosporia: Effects on growth and parasitism

The biocontrol potential of Pochonia chlamydosporia, a fungus with parasitic activity against economically important plant-parasitic nematodes, can be influenced by abiotic factors such as water availability. The objective of this study was to evaluate the effects of different water stress regimes on in vitro growth, sporulation, germination and parasitism of P. chlamydosporia isolates. The osmotic water potential of 1.7% corn meal agar (CMA) was modified by addition of potassium chloride (KCl) or glycerol, and the matric water potential was modified using polyethylene glycol (PEG 8000). The fungus was able to grow over a range of potentials but radial growth rates decreased with the increase of osmotic and matric stress. No growth was observed at −10 MPa on 1.7% CMA amended with glycerol and at −7.1 MPa on medium with PEG 8000 but all isolates were able to resume growth when transferred onto unmodified 1.7% CMA. The production of chlamydospores was repressed in both osmotic and matric modified media. Although the production of conidia increased in medium modified with KCl, the germination rate was lower. Spores/hyphal fragments remained viable in all isolates that were previously inoculated onto media with growth-limiting water potential (−10 MPa on 1.7% CMA amended with glycerol and −10 MPa on medium with PEG 8000). The percentage of viable conidia produced on 1.7% CMA, after inoculation under osmotic or matric stress conditions for 25 days, was over 74.5% in all isolates (osmotic stress) and ranged from 1% (Pc1) to 65.8% (Pc280) (matric stress). The in vitro infection of potato cyst nematodes, Globodera rostochiensis eggs by P. chlamydosporia isolates, grown under these limiting conditions, was studied using a standard bioassay. The percentage of parasitized eggs was significantly higher under osmotic stress except for isolates Pc2 and Pc3. P. chlamydosporia spores/hyphal fragments can remain viable at water potentials limiting for growth, for prolonged periods of time, suggesting that the osmoregulation mechanisms, used to compensate water stress, affect in vitro sporulation and increased pathogenicity. Knowledge on water requirements of P. chlamydosporia enables a better understanding of its survival and growth strategies in the soil environment and could aid the development of effective strategies to increase the production and quality of inoculum, thus contributing to the implementation of biosafe, sustainable management strategies against plant-parasitic nematodes.     

Water potential affects Coniothyrium minitans growth, germination and parasitism of Sclerotinia sclerotiorum sclerotia

Water availability is an important environmental factor which has major effects on fungal activity. The effects of osmotic (KCl amended agar) and matric Polyethylene glycol ((PEG) 8000 amended agar) potentials over the range -0.1 to -5.0MPa on mycelial growth and conidial germination of eight isolates of the sclerotial parasite Coniothyrium minitans was assessed. The influence of soil water potential on the ability of three selected isolates (LU112, LU545, and T5R42i) to parasitise sclerotia of the plant pathogen Sclerotinia sclerotiorum was determined. For all eight C. minitans isolates, decreasing osmotic and matric potentials caused a reduction in mycelial growth and conidial germination. Isolates were more sensitive to decreasing matric potential than osmotic potential. Across the isolates, growth at an osmotic potential of -5.0MPa was 30-70% of the growth seen in the control, whereas less than 20% of the control growth was seen at the corresponding matric potential. Across all isolates no conidial germination was seen at matric potential of -5.0MPa. The C. minitans isolates varied in their sensitivity to decreasing water potentials. Mycelial growth and conidial germination of three isolates (LU112, Conio, and CH1) were more tolerant of low osmotic potential and matric potential with respect to mycelial growth. Isolates T5R42i and LU430 were least tolerant. In contrast, conidial germination of isolates Conio, LU545, and T5R42i were less sensitive to decreasing matric potential. Soil water potential was seen to affect infection and viability of sclerotia by the three C. minitans isolates. Isolate LU545 reduced sclerotial viability over a wider water potential range (-0.01 to -1.5MPa) compared with LU112 (-0.01 to -1.0MPa), with isolate T5R42i being intermediate. Indigenous soil fungi (Trichoderma spp. and Clonostachys rosea) were recovered from sclerotia but did not result in reduction in sclerotial viability. The relevance of these results in relation to biocontrol activity of C. minitans in soil is discussed.     

Impact of osmotic and matric water stress on germination, growth, mycelial water potentials and endogenous accumulation of sugars and sugar alcohols in Fusarium graminearum

Studies were conducted to determine the effect of osmotic (NaCl, glycerol) and matric (PEG 8000) water stress on temporal germination and growth of two F. graminearum strains over the water potential range of -0.7 to -14.0 MPa at 15 and 25 C. The effect on endogenous water potentials and accumulation of sugars and sugar alcohols also were measured. For both strains, germination occurred rapidly over the same range of osmotic or matric potential of -0.7 to -5.6 MPa after 4-6 h incubation. At lower osmotic and matric potentials (-7.0 to -8.4 MPa), there was a lag of up to 24 h before germination. Optimum germ-tube extension occurred between -0.7 and -1.4 MPa for both strains but varied with the solute used. Growth was optimal at -1.4 MPa and 25 C in response to matric stress, with the minimum being about -8.0 and -11.2 MPa at 15 and 25 C, respectively. In contrast, F. graminearum grew fastest at -0.7 MPa and was more tolerant of solute stress modified with either glycerol or NaCl with a minimum of about -14.0 MPa at 15 and 25 C. A decrease in the osmotic/matric water potential of the media caused a large decrease in the mycelial water potential (Ψ(c)) as measured by thermocouple psychrometry. In general, the concentration of total sugar alcohols in mycelia increased as osmotic and matric potential were reduced to -1.2 MPa. However, this increase was more evident in mycelia from glycerol-amended media. The quality of the major sugar alcohol accumulated depended on the solute used to generate the water stress. The major compounds accumulated were glycerol and arabitol on osmotically modified media and arabitol on matrically modified media. In response to matric stress, the concentration of trehalose in colonies generally was higher in the case of osmotic stress. In each water-stress treatment there was a good correlation between Ψ(c) and total sugar alcohol content.     

The importance of water potential range tolerance as a limiting factor on Trichoderma spp. biocontrol of Sclerotinia sclerotiorum

Biological control of fungal phytopathogens is often more variable in efficacy compared with disease suppression achieved by conventional pesticide use. Matching the environmental range of a potential biocontrol agent with that of the target phytopathogen is necessary if consistent disease suppression is to be achieved under field conditions. Strains of Trichoderma that could parasitise sclerotia of Sclerotinia sclerotiorum had their spore germination and mycelial growth (five strains) and ability to parasitise sclerotia (two strains) tested under a range of water potentials under laboratory conditions. Relative mycelial growth and germination of all strains decreased with decreasing osmotic and matric potentials, with matric potential having a greater impact on growth and germination over the range examined. Trichoderma harzianum LU698 mycelial growth was the least affected by decreasing osmotic potential than the other isolates, and Trichoderma atroviride LU141 growth least affected by decreasing matric potential. The germination of LU698 and LU144 was also generally less affected by decreasing osmotic potential, although generally decreasing matric potential had the greatest affect on the germination of LU698 along with T. atroviride LU132. Soil treatments of LU698 and Trichoderma asperellum LU697 reduced sclerotial viability in all but the lowest soil water potential tested, with LU698 being most effective at ?0.1 and ?0.3 MPa after 28 days and LU697 most effective at ?0.01 and ?1.5 MPa after 28 days. We conclude that differences in the tolerance of potential biocontrol agents to changing water potential is an important experimental factor to consider when assaying biocontrol or making predictions of biocontrol efficacy in the field.     

Effect of natural maize phytochemicals on Aspergillus section Flavi sclerotia characteristics under different conditions of growth media and water potential

The effects of the natural phytochemicals trans-cinnamic acid (CA) and ferulic acid (FA) at concentrations of 1–20 mM (CA) and 1–25 mM (FA) on sclerotial production by Aspergillus flavus and Aspergillus parasiticus were evaluated. Studies on sclerotium number and size were carried out in different growth media and water potentials (MPa). High concentrations of CA (20 mM, ?0.75 MPa; 10 mM, ?3.5 MPa) and FA (10, 20, 25 mM, ?0.75 and ?3.5 MPa) significantly reduced sclerotial production of Aspergillus strains. Overall, CA at concentrations of 10 and 20 mM on Czapek Dox medium (CD), maize meal extract agar (MMEA) and maize meal extract agar with sucrose and NaNO₃ (MMEA S/N) inhibited sclerotium most in the four species assayed. The data show that the sclerotia characteristics of A. flavus and A. parasiticus were influenced by natural phytochemicals and modifications of growth media and water potential. CA and FA could be used at high concentrations to prevent the survival of Aspergillus species in grain.     

Effect of solute and matric potential on in vitro growth and sporulation of strains from a new population of Aspergillus flavus isolated in Italy

The effect of temperature and different solute (Ψ_s) and matric potentials (Ψ_m) on growth and sporulation of three aflatoxigenic strains of Aspergillus flavus isolated from contaminated maize in northern Italy was determined. The Ψ_s of maize-based media were modified ionically (NaCl) and non-ionically (glycerol) and the Ψ_m with PEG 8000 in the range −1.4 to −21.0 MPa at 25 and 30 °C. Both temperature and Ψ_s/Ψ_m stress had statistically significant effects on growth rates of the three strains. Faster growth occurred at 30 °C and −1.4 and −2.8 MPa. A. flavus strains were more sensitive to Ψ_m than Ψ_s stress with limits of −9.8 MPa and −14 to−18 MPa, respectively. Sporulation was significantly influenced by Ψ_s potential, solute type and temperature. This suggests that these aflatoxigenic strains of A. flavus isolated from aflatoxin-contaminated maize are probably able to colonise crop debris rapidly at prevailing temperatures and water stress conditions. This type of information on the ecology of aflatoxin producing A. flavus strains isolated in Italy will contribute to the development of a systems model to predict their activity in crop residue and colonisation of maize grain.     

Ecophysiological requirements on seed germination of a Mediterranean perennial grass (Stipa tenacissima L.) under controlled temperatures and water stress

Stipa tenacissima L. (alpha grass) steppes are one of the most representative ecosystems in arid Mediterranean ecosystems. On the one hand these steppes, which are perpetually exposed to climate and strong anthropogenic pressure, have undergone severe degradation. On the other hand, the ability of S. tenacissima to regenerate naturally is significantly reduced. In this study the germination response and seedling emergence of S. tenacissima are examined in relation to the main environmental factors (water stress and temperature) under laboratory-controlled conditions. The main aim of this paper was to investigate the influence of temperature over a temperature range (10 °C–30 °C) and water stress induced by the solutions of polyethylene glycol (PEG)-6000 (0 to − 1.6 MPa) for a period of 30 days, on the germination behavior of S. tenacissima seeds. The results showed that temperatures between 10° and 20 °C seem to be favorable for the germination of this species, with optimum temperatures among accessions found in 20 °C. When seeds were water-stressed, germination severely decreased at − 0.8 MPa, indicating that the accession resistance limits to the water stress, and was completely inhibited at − 1.6 MPa. Consequently, the final germination percentage (FGP) decreased and the mean time germination (MTG) increased. Based on the empirical data of the germination rate, we estimated that the parameters of the thermal time and hydrotime models showed different values in all accessions which proves the difference between accession adaptive capacities.     

Seed storage and germination in Kumara plicatilis,a tree aloe endemic to mountain fynbos in the Boland,south-western Cape,South Africa

Seed storage under appropriate conditions is a relatively inexpensive means of safeguarding plant genetic material for ex situ conservation. Post-storage germination trials are used to determine the viability of stored seeds, and hence the efficacy of the particular storage treatment. Kumara plicatilis (= Aloe plicatilis) is a tree aloe endemic to mountain fynbos in the Boland, south-western Cape. The viability and germination behaviour of K. plicatilis seeds were assessed for seeds stored for four and nine months at − 80 °C, 4 °C, 25 °C and under ambient conditions in a laboratory. Seeds were germinated under controlled conditions and germination rates and percentages determined. Ungerminated seeds were tested for viability using tetrazolium salt. Seed viability was not significantly reduced during storage. Seeds stored at − 80 °C for four and nine months exhibited the fastest germination rate overall (both 5.9 ± 0.3 weeks, mean ± S.E.), and slowest was for seeds stored under ambient conditions for four and nine months (both 7.8 ± 0.4 weeks). All seed lots showed similar percentage germination after four months of storage (78.0–90.4%). The highest percentage germination overall was for seeds stored at − 80 °C for four months (90.4%) and the lowest was for seeds kept at 4 °C and − 80 °C for nine months (39.2 and 39.6%, respectively). Respective percentage viability for ungerminated seeds in these two treatments was 82% and 87%, respectively, indicating the induction of secondary dormancy. Induced dormancy triggered by protracted cold temperatures may be an adaptation that enables seeds to survive prolonged extreme conditions that are unfavourable for germination. Further research on the long-term storage of aloe seeds would be beneficial for developing long-term seed storage and germination testing protocols for ex situ conservation.     

Cultural studies of Morchella elata

The in vitro growth of Morchella elata was characterized with respect to the effects of a variety of substrates, isolates, developmental status of the parental ascoma, temperature, and pH. Optimal substrates for growth included sucrose, mannose and lactose, but the growth of some isolates was substantially reduced in some composite media. Maltose and potato-dextrose media limited growth and caused changes in colony morphology; mycelial pigmentation was black in the case of maltose, and mycelial margins were plumose in potato-dextrose cultures. Rapid growth was most reliably achieved in a composite medium containing 1:1 sucrose:mannose. Isolates derived from single ascospores shortly after ejection from ascomata varied in ability to grow in the various substrates. This may be related to variable maturity or dormancy; increasing growth rates correlated with pileus length in the parental ascomata, and ascomata that initially produced slower-growing or abortive colonies produced faster-growing colonies after storage at 20 °C for 96 wk. The growth of M. elata derived from recently ejected ascospores was optimal at 16–24 °C or above for a faster-growing isolate, and 20–24 °C or above for a slow-growing isolate. Although neither isolate grew at 8 °C or below in an initial experiment, spawn cultured on puffed wheat at 28 °C produced mycelia that proliferated when transferred to soil media and incubated at 8 °C. Growth of M. elata in liquid cultures adjusted with potassium hydroxide was optimal at pH 7.0, and was relatively sensitive to more acidic or alkaline pH. When calcium carbonate was used to adjust pH, optimal growth shifted to pH 7.7 or above, suggesting that wood ash and other calcium compounds may not only stimulate growth in natural settings, but also alter the optimal pH for proliferation of M. elata. Further studies with other substrate combinations and incubation conditions will be necessary to fully understand the connections between in vitro growth and the ecological behaviour of the fungus.     

Characterization and efficacy of Muscodor cinnamomi in promoting plant growth and controlling Rhizoctonia root rot in tomatoes

Muscodor cinnamomi was selected and investigated for its in vitro ability to produce indole-3-acetic acid (IAA) to solubilize different toxic metal (Ca, Co, Cd, Cu, Pb, Zn)-containing insoluble minerals and tolerance to metals, herbicides and an insecticide. The results indicated that this fungus is able to produce IAA (45.36 ± 2.40 μg ml⁻¹) in liquid media. This phytohormone stimulated coleoptile elongation, and increased seed germination and root elongation of tested plants. The metal tolerance and solubilizing ability depended on the type of insoluble minerals. M. cinnamomi showed the highest growth tolerance on Ca-containing media at 150 mM, followed by Zn-containing media at 100 mM and Cd-containing media at 10 mM. This fungus tolerated the three herbicides (2,4-d-dimethylammonium, glyphosate and paraquat dichloride) and an insecticide (methomyl) at the recommended dosages for field application. Moreover, M. cinnamomi completely controlled Rhizoctonia solani AG-2 root rot in tomato plants, and increased root length, shoot dry weight and root dry weight. This is the first report of in vitro IAA production, solubilization of insoluble metal minerals, and tolerance to herbicides, an insecticide and metals as well as the plant growth promoting ability of M. cinnamomi.     

Effect of high salinity on Atriplex portulacoides: Growth,leaf water relations and solute accumulation in relation with osmotic adjustment

Atriplex (Halimione) portulacoides is a halophyte with potential interest for saline soil reclamation and phytoremediation. Here, we assess the impact of salinity reaching up to two-fold seawater concentration (0–1000 mM NaCl) on the plant growth, leaf water status and ion uptake and we evaluate the contribution of inorganic and organic solutes to the osmotic adjustment process. A. portulacoides growth was optimal at 200 mM NaCl but higher salinities (especially 800 and 1000 mM NaCl) significantly reduced plant growth. Na⁺ and Cl⁻ contents increased upon salt exposure especially in the leaves compared to the roots. Interestingly, no salt-induced toxicity symptoms were observed and leaf water content was maintained even at the highest salinity level. Furthermore, leaf succulence and high instantaneous water use efficiency (WUE_i) under high salinity significantly contributed to maintain leaf water status of this species. Leaf pressure–volume curves showed that salt-challenged plants adjusted osmotically by lowering osmotic potential at full turgor (Ψ_π¹⁰⁰) along with a decrease in leaf cell elasticity (values of volumetric modulus elasticity (ε) increased). As a whole, our findings indicate that A. portulacoides is characterized by a high plasticity in terms of salt-response. Preserving leaf hydration and efficiently using Na⁺ for the osmotic adjustment especially at high salinities (800–1000 mM NaCl), likely through its compartmentalization in leaf vacuoles, are key determinants of such a performance. The selective absorption of K⁺ over Na⁺ in concomitance with an increase in the K⁺ use efficiency also accounted for the overall plant salt tolerance.     

Antifungal potentiality of mycogenic silver nanoparticles capped with chitosan produced by endophytic Amesia atrobrunnea

This research reports the fabrication of silver nanoparticles (AgNPs) from endophytic fungus, Amesia atrobrunnea isolated from Ziziphus spina-christi (L.). Influencing factors for instance, thermal degree of incubation, media, pH, and silver nitrate (AgNO₃) molarity were optimized. Then, the AgNPs were encapsulated with chitosan (Ch-AgNPs) under microwave heating at 650 W for 90 s. Characterization of nanoparticles was performed via UV–visible (UV–vis) spectrophotometer, Fourier-transform infrared spectrophotometer (FTIR), zeta potential using dynamic-light scattering (DLS), and field-emission-scanning electron microscope (FE-SEM). Anti-fungal activity of Ch-AgNPs at (50, 25, 12.5, 6.25 mg/L) was tested against Fusarium oxysporum, Curvularia lunata, and Aspergillus niger using the mycelial growth inhibition method (MGI). Results indicated that Czapek-dox broth (CDB) with 1 mM AgNO₃, an acidic pH, and a temperature of 25–30 °C were the optimum for AgNPs synthesis. (UV–vis) showed the highest peak at 435 nm, whereas Ch-AgNPs showed one peak for AgNPs at 405 nm and another peak for chitosan at 230 nm. FTIR analysis confirmed that the capping agent chitosan was successfully incorporated and interacted with the AgNPs through amide functionalities. Z-potential was −19.7 mV for AgNPs and 38.9 mV for Ch-AgNPs, which confirmed the significant stability enhancement after capping. FES-SEM showed spherical AgNPs and a reduction in the nanoparticle size to 44.65 nm after capping with chitosan. The highest mycelial growth reduction using fabricated Ch-AgNPs was 93% for C. lunata followed by 77% for A. niger and 66% F. oxysporum at (50 mg/L). Biosynthesis of AgNPs using A. atrobrunnea cell-free extract was successful. Capping with chitosan exhibited antifungal activity against fungal pathogens.     

Effects of osmotic and matric potential on radial growth and accumulation of endogenous reserves in three isolates of Pochonia chlamydosporia

For the first time, the effects of varying osmotic and matric potential on fungal radial growth and accumulation of polyols were studied in three isolates of Pochonia chlamydosporia. Fungal radial growth was measured on potato dextrose agar modified osmotically using potassium chloride or glycerol. PEG 8000 was used to modify matric potential. When plotted, the radii of the colonies were found to grow linearly with time, and regression was applied to estimate the radial growth rate (mm day^?1). Samples of fresh mycelia from 25-day-old cultures were collected and the quantity (mg g^?1 fresh biomass) of four polyols (glycerol, erythritol, arabitol and mannitol) and one sugar (glucose) was determined using HPLC. Results revealed that fungal radial growth rates decreased with increased osmotic or matric stress. Statistically significant differences in radial growth were found between isolates in response to matric stress (P<0.006) but not in response to osmotic stress (P=0.759). Similarly, differences in the total amounts of polyols accumulated by the fungus were found between isolates in response to matric stress (P<0.001), but not in response to osmotic stress (P=0.952). Under water stress, the fungus accumulated a combination of different polyols important in osmoregulation, which depended on the solute used to generate the stress. Arabitol and glycerol were the main polyols accumulated in osmotically modified media, whereas erythritol was the main polyol that was accumulated in media amended with PEG. The results found that Pochonia chlamydosporia may use different osmoregulation mechanisms to overcome osmotic and matric stresses.     

Production of 1,3-propanediol, 2,3-butanediol and ethanol by a newly isolated Klebsiella oxytoca strain growing on biodiesel-derived glycerol based media

The production of 1,3-propanediol, 2,3-butanediol and ethanol was studied, during cultivations of strain Klebsiella oxytoca FMCC-197 on biodiesel-derived glycerol based media. Different kinds of glycerol feedstocks and experimental conditions had an important impact upon the distribution of metabolic products; production of 1,3-propanediol was positively influenced by stable pH conditions and by the absence of N₂ gas infusions throughout the fermentation. Thus, during batch bioreactor fermentations conducted at increasing glycerol concentrations, 1,3-propanediol at 41.3 g/L and yield ～47% (w/w) was achieved at initial glycerol concentration ～120 g/L. At even higher initial glycerol media (150 and 170 g/L), growth was not ceased, but 1,3-propanediol production declined. During fed-batch fermentation under optimal experimental conditions, 126 g/L of glycerol were converted into 50.1 g/L of 1,3-propanediol. In this experiment, also 25.2 g/L of ethanol (conversion yield ～20%, w/w) were formed. A batch-bioreactor culture was performed under non-sterilized conditions and the 1,3-propanediol production was almost equivalent to the sterilized process. Concerning 2,3-butanediol formation, the most detrimental parameter was the absence of N₂ sparging and as a result, no 2,3-butanediol was produced. The presence of glucose as co-substrate seriously enhanced 2,3-butanediol production; when commercial glucose was employed as sole substrate, 32.1 g/L of 2,3-butanediol were formed.     

Effects of temperature,pH and water potential on growth of four fungi with disease biocontrol potential

Effects of temperature, pH and water potential on blomass production or hyphal extension of Gliocladium virens (G20) and three Trichoderma isolates were determined in vitro. Optimum blomass production occurred between 20 and 30°C and at pH ranges between 4.6 and 6.8. Two isolates of T. viride grew at 5°C and G. virens grew at 35°C but no isolates grew at 40°C. Hyphal extension rates and conidial germination of all fungi declined with decreasing water potential over the range -0.7 to -14.0 MPa. In general, growth rates for each isolate were lower on potato/dextrose agar with water potential adjusted with polyethylene glycol than when adjusted with NaCl or glycerol. No mycelial growth or spore germination occurred on agar at-14.0 MPa.The authors are with the Microbiology and Crop Protection Department, Horticulture Research International, Littlehampton, West Sussex BN17 6LP, UK. J.M. Whipps is the corresponding author     

Elicitation with abiotic stresses improves pro-health constituents,antioxidant potential and nutritional quality of lentil sprouts

Phenolic content and antioxidant potential of lentil sprouts may be enhanced by treatment of seedlings in abiotic stress conditions without any negative influence on nutritional quality.The health-relevant and nutritional quality of sprouts was improved by elicitation of 2-day-old sprouts with oxidative, osmotic, ion-osmotic and temperature stresses. Among the sprouts studied, those obtained by elicitation with osmotic (600 mM mannitol) and ion-osmotic (300 mM NaCl) shocks had the highest total phenolic content levels: 6.52 and 6.56 mg/g flour, respectively. Oxidative stress significantly enhanced the levels of (+)-catechin and p-coumaric acid. A marked elevation of the chlorogenic and gallic acid contents was also determined for sprouts induced at 4 °C and 40 °C. The elevated phenolic content was translated into the antioxidant potential of sprouts, especially the ability to reduce lipid oxidation. A marked elevation of this ability was determined for seedlings treated with 20 mM, 200 mM H₂O₂ (oxidative stress) and 600 mM mannitol (osmotic stress); about a 12-fold, 8-fold and 9.5-fold increase in respect to control sprouts. The highest ability to quench free radicals was observed in sprouts induced by osmotic stress (IC₅₀- 4.91 and 5.12 mg/ml for 200 mM and 600 mM mannitol, respectively). The highest total antioxidant activity indexes were determined for sprouts elicited with 20 mM H₂O₂ and 600 mM mannitol: 4.0 and 3.4, respectively. All studied growth conditions, except induction at 40 °C, caused a significant elevation of resistant starch levels which was also affected in a subsequent reduction of starch digestibility.Improvement of sprout quality by elicitation with abiotic stresses is a cheap and easy biotechnology and it seems to be an alternative to conventional techniques applied to improve the health promoting phytochemical levels and bioactivity of low-processed food.     

Bacillus spp. and Pseudomonas putida as inhibitors of the Colletotrichum acutatum group and potential to control Glomerella leaf spot

The control of Glomerella leaf spot (GLS) in Brazil is solely based on fungicide sprays and new alternatives are needed. In apple, few biological control methods have been evaluated, and most have focused on post-harvest pathogens. Therefore, the objectives of this work were to study the mode of action of three bacterial strains and the commercial product Serenade® (Bacillus subtilis) against the Colletotrichum acutatum group, the causal agents of GLS, and to evaluate the influence of bacterial isolates and Serenade® on the development of the first cycle of infection disease under controlled conditions. To assess the mode of action of the bacterial isolates against strains of the C. acutatum group, in vitro tests were performed. It was tested the effect of the bacteria on conidial germination and mycelial growth, using three methodologies, (i) fungal-bacterial co-cultivation, (ii) bacterial thermostable metabolites and (iii) bacterial volatile compounds. The influence of the bacterial isolates on the GLS development was assessed using apple seedlings. The seedlings were first sprayed weekly with bacterial suspension for 5 weeks, and were then inoculated with conidia suspensions (10⁴ conidia mL⁻¹) of C. acutatum group isolates. Seedlings were maintained in chambers (CONVIRON) at 25 °C and a 12-h light regime. Disease severity of GLS was evaluated daily by counting typical lesions caused by C. acutatum group on all leaves during 12 consecutive days. The disease progress curve was fitted to nonlinear models for incidence and severity data. The treatments were compared by contrasting epidemiological parameters. Bacillus sp. isolated from the apple phylloplane inhibited more than 60% of the C. acutatum group conidial germination. The mode of action of Bacillus sp. and Bacillus alcalophilus on the C. acutatum group was through the production of fixed and volatile compounds, which inhibited mycelial growth. The primary mode of action of Serenade® on the C. acutatum group was the production of thermostable metabolites capable of completely inhibiting mycelial growth. In the GLS disease cycle, it was possible to adjust the monomolecular model for incidence and the number of lesions. There were significant differences between the epidemiological parameters of GLS in seedlings treated with apple phylloplane bacteria or with Serenade® as compared to the controls, indicating a potential for the use of biological control to manage GLS in apple orchards.     

Frequency of Continuous Glucose Monitoring use and Change in Hemoglobin A1C for Adults With Type 1 Diabetes in a Clinical Practice Setting

ObjectiveTo estimate the frequency of continuous glucose monitoring (CGM) use and change in hemoglobin A1c (HbA1c) compared to self-monitoring of blood glucose (SMBG) alone in adults with type 1 diabetes in a clinical practice setting.MethodsWe retrospectively identified 66 adult type 1 diabetes patients at the Barbara Davis Center for Diabetes (BDC) who first initiated CGM between 2006 and 2011 and 67 controls using SMBG. The frequency of CGM use was estimated from survey recall and defined as the mean number of days/month of CGM use during a maximum follow-up of 10 months. Change in HbA1c was calculated as the difference between the baseline value and the lowest follow-up value.ResultsThe mean change in HbA1c for CGM users was − 0.48% (95% confidence interval [CI]: − 0.67, − 0.28) and for SMBG users was − 0.37% (95% CI: − 0.56, − 0.18). The between-group mean difference in change in HbA1c, adjusted for patient characteristics, was − 0.11% (95% CI: − 0.38, 0.16), whereas the subgroup with a baseline HbA1c ≥ 7.0% and users of CGM ≥ 21 days/month was − 0.36% (95% CI, − 0.78, 0.05). Nearly half (n = 32, 48%) used CGM < 21 days/month. The reasons for low frequency of CGM use or discontinuation included sensor costs, frequency of alarms, inaccuracy, and discomfort.ConclusionsThese CGM data from clinical practice suggest a trend toward decreasing HbA1c for adults with type 1 diabetes, especially in patients with higher baseline HbA1c and higher frequency of CGM use. Future studies are needed to assess the use of CGM in larger populations of clinical practice adult type 1 diabetes patients. (Endocr Pract. 2014;20:1007-1015)     

Dynamics of tear fluid components transportation through contact lenses

The interference optical method has been applied to monitor the transportation of Na⁺ and Cl⁻ in solution through new and used contact lenses. The phenomenon of passive transportation (simple diffusion), induced by differences in osmotic pressure on both sides of the contact lenses has been discussed. Permeability coefficient of contact lenses of different optical power: −2.75 D and −3.75 D has been calculated.     

A reassessment of the prevalent organic solutes constitutively accumulated and potentially involved in osmotic adjustment in pear leaves

In higher plants, osmotic adjustment at the various levels of plant organization is partly achieved through accumulation of a range of osmolytes especially LMW organic solutes often termed as osmotic solutes. A metabolite profiling of crude extracts of mature pear leaves of a range of 8 Pyrus genotypes was performed using current HPLC, UPLC and ¹H NMR spectroscopy techniques in order to identify such putative compounds. Using as variables the concentrations of 45 identified substances and those of a restricted number of unknowns, all belonging to LMW carbohydrates, polyols, organic acids, amino acids and phenolics on the one hand, and the varieties investigated as individuals on the other, we generated a set of data analyzed further by PCA. Those varieties were discriminated into three clusters respectively comprised of the four Asian varieties, the European variety Williams grafted onto 4 different rootstocks, and the two other European varieties Conference and Angelys. These metabolic phenotypes were shown to rely more on scion genotypes than on rootstocks. High to very high amounts of sorbitol (average content of 363 μmol g^?1 DW) associated with low amounts of mannitol and myo-inositol were found in all genotypes as well as in a local ecotype of P. communis where the hexitol accounted for 7.3% DW. Sorbitol actually represented up to 30-40% of the total osmotically active organic solutes accumulated in the set of pear leaves investigated, and it was shown to be significantly more abundant in the variety Williams than in Asian ones (p < 0.01). In contrast, the other well-known compatible solute glycine betaine, barely detectable using ¹H NMR spectroscopy or HPLC, occurred in leaves of all pear varieties at weak levels lower than 2 μmol g^?1 DW which suggested a minor role in osmotic adjustment. Its amount does not seem to be altered in response to an osmotic upshift applied to detached leaves or to depend on exogenously supplied ABA. For non-sustantiated reasons, these results are in contrast with those showing elsewhere very high accumulation of GB in the Asian genotype Su li. In this study mature leaves of this genotype collected from the same tree in July 2007 and July 2006 were shown to contain respectively 0.59 ± 0.04 and 0.85 ± 0.04 μmol g^?1 DW. Other abundant organic substances like arbutine, quinic acid, malic acid, sucrose as well as chlorogenic acid and other quinic acid adducts, might also behave as osmotically active substances. In addition to arbutine, its derivative hydroquinone, chlorogenic acid and structurally related substances might be involved in protective functions against secondary oxidative stresses induced by abiotic and biotic stresses encountered during the growing season.