Effect of water activity and temperature on germination and growth of Penicillium digitatum,P. italicum and Geotrichum candidum

AIMS: This study compares the effect of temperature (4-37 degrees C) and water activity (aw: 0.99-0.87) and their interactions on the germination rates, lag times prior to germination and mycelial growth 'in vitro' of Penicillium digitatum, P. italicum and Geotrichum candidum, the main postharvest pathogens affecting citrus fruits. METHODS AND RESULTS: Germination and growth were markedly influenced by temperature and aw. Generally, lag times were longer and germination and growth rates were slower when conditions of temperature and aw were far from optimum. All the studied species were able to germinate over a range of 4-30 degrees C at 0.995 aw, although in non-optimal conditions P. digitatum only reached 40-60% of germinated conidia. At low temperatures, P. italicum germinated and grew faster than P. digitatum and G. candidum, particularly at 0.95 aw. Penicillium italicum was also able to germinate and grow in the driest studied conditions (0.87 aw), while G. candidum did not germinate under 0.95 aw. CONCLUSIONS: Knowledge of the ecological requirements of these fungi is important in order to understand their behaviour in natural situations and to predict fungal spoilage on citrus fruits.     

Suppression and promotion of growth by ethylene in rice seedlings depends on ambient humidity

We examined the effect of ethylene on the growth of rice seedlings (Oryza sativa L.) at various degrees of humidity. Ethylene significantly suppressed the growth of shoots when applied to seedlings grown under 30% relative humidity (RH), but promoted the growth of shoots when applied to seedlings grown under 100% RH. The application of gibberellic acid (GA₃) promoted the elongation of shoots in seedlings grown under 30% and 100% RH. Ethylene inhibited the shoot elongation induced by GA₃ at 30% RH, but enhanced the elongation induced by GA₃ at 100% RH. These results indicate that ethylene can either promote or suppress the growth of rice shoots depending on ambient humidity, and that these actions of ethylene may be mediated through modulating the responsiveness of shoots to gibberellin.     

Separate effects of moisture content and water activity on the hyphal extension of Penicillium rubens on porous media

To prevent indoor fungal growth, understanding the moisture relations of fungi is a key element. Indoor moisture is quantified by the relative humidity (RH). RH controls the water activity of the indoor materials that fungi grow on, a well‐studied parameter known to limit fungal growth. RH, however, also controls the amount of water present in these materials, the moisture content. The significance of the moisture content of these materials to indoor fungal growth is currently overlooked. In the work reported here, growth experiments with the indoor fungus Penicillium rubens on gypsum substrates were performed to test whether the moisture content influences growth on porous materials. Second, we report the development of a video microscopy method that for the first time quantified hyphal growth on a porous material. It is found that a higher moisture content leads to earlier colonization and higher hyphal extension rates. This is a fundamental step in unravelling the effect of RH on indoor fungal growth. The real‐time monitoring of colonization of gypsum provides a new view of growth on indoor surfaces.     

Effect of the Urtica dioica agglutinin on germination and cell wall formation of Phycomyces blakesleeanus Burgeff

The lectin from stinging nettle rhizomes, Urtica dioica agglutinin (UDA), did not affect the evolution of wet and dry weight, protein, nucleic acid, ATP, cAMP and glycerol content during early germination of Phycomyces blakesleeanus spores. However, earlier investigations established a strongly reduced mycelial growth of several phytopathogenic fungi by this small plant lectin. Total uptake and incorporation of radioactive precursors showed no differences between UDA or control hyphae, but UDA significantly altered the distribution patterns of [¹⁴C]-glucose incorporated into the walls of Phycomyces blakesleeanus (more label was recovered in the chitin fraction). Moreover, a small but significant stimulation of chitin synthase and a similar inhibition of chitin deacetylase was found in cell wall preparations. These observations could lead to a better understanding of plant-pathogen interrelationships and to a further elucidation of cell wall structure in fungi.Abbreviations GlcNAc N-Acetylglucosamine - PDB potato dextrose broth - PMM Phycomyces minimal medium - UDA Urtica dioica agglutinin - TEA tri-ethyl-amine - DAB 1,4-diaminobutanone     

The effects of temperature,water activity and pH on the growth of Aeromonas hydrophila and on its subsequent survival in microcosm water

AIMS: The influence of temperature, water activity and pH on the growth of Aeromonas hydrophila, and on its survival after transfer in nutrient-poor water were assessed. METHODS AND RESULTS: Experiments were carried out according to a Box-Behnken matrix at 10-30 degrees C, 0.95-0.99 water activity (aw) and pH 5-9. The effect of each factor on the kinetic parameters of growth (i.e. the maximal specific growth rate, mumax, and the lag time, lambda) and on the decline of the bacteria in microcosm water (time to obtain a reduction of 5 log, T5 log) were studied by applying central composite design. CONCLUSIONS: The major effect of temperature and water activity on the growth of A. hydrophila was highlighted, whereas the effect of pH in these experimental conditions was not significant. Models describing the effect of environmental parameters on the growth of A. hydrophila were proposed. The effect of the growth environment, and particularly the incubation temperature, have an influence on the survival ability of the bacteria in nutrient-poor water. SIGNIFICANCE AND IMPACT OF THE STUDY: The Box-Behnken design was well suited to determine the influence of environmental factors on the growth of A. hydrophila and to investigate the effect of previous growth conditions on its survival in microcosm water.     

Influence of inoculum preparation on the growth of Penicillium chrysogenum

AIMS: The influence of the spore preparation on subsequent fungal growth of Penicillium chrysogenum was assessed. METHODS AND RESULTS: The influence of four factors [the nature of the diluting solution (physiological water and physiological water added with Tween-80), the age of the sporulating culture (4, 8 and 12 days), the strain (737, 738 and 740) and the inoculum size (102, 103, 104 and 105 spores ml(-1)] on two responses (i.e. the radial growth rate, mu, and the lag time, lambda) was studied using an experimental screening methodology. CONCLUSIONS: The main conclusion was the strong effect of the inoculum size on lambda. In contrast, the diluting solution had no effect on both the experimental responses. In order to obtain the highest growth rates, it is recommended to use 4-day-old sporulating cultures with an inoculum size of 102 spores ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: There is a need for standardizing spore preparation in predictive mycology. The screening methodology is a powerful tool to determine the influence of qualitative and quantitative factors on various biological responses and can be applied widely in microbiology.     

板蓝根种子的吸水特性及发芽条件研究

采用室内测定方法,对板蓝根种子的吸水特性及不同处理下种子的发芽率、发芽势和相对活力指数等进行比较研究。结果表明,板蓝根种子具有快速吸水的生物学特性,分为快速吸水期、缓慢吸水期和缓慢吐水期三个阶段,入水后2 h的吸水量达最大吸水量的73.29%,为快速吸水阶段;温度对板蓝根种子的萌发影响较大,最适发芽温度为30℃。光照、发芽床和种子预处理对板蓝根种子的萌发无明显影响。这说明板蓝根种子的萌发主要取决于水分和温度。     

Hydro- and thermotimes for conidial germination kinetics of the ochratoxigenic species Aspergillus carbonarius in vitro,on grape skin and grape flesh

The objective was to compare the ability of spores of Aspergillus carbonarius to germinate in vitro, in situ on grape skin and grape flesh in relation to temperature (15–40 °C) and different relative humidities (100–85 % RH). Spores were inoculated as a spore suspension (10⁶ spores ml⁻¹) onto the surface of white organic grapes and directly onto cut grape flesh. For comparison, spores were spread plate onto a synthetic grape juice medium (SGM) modified to the equivalent water activity (a_w) range of 0.995–0.85. This showed that conidia germinated more rapidly on grape flesh (6 h) followed by that on the SGM medium (9 h) and then grape skin (24 h) under optimal condition of 30–35 °C and 100 % RH. At marginal conditions, such as 15 °C and 85–90 % RH, germination was very slow. The time to 5 % germination was significantly shorter on grape flesh than in vitro on grape medium and slowest on grape skin. This suggests that damaged grapes provide the main method of infection and contamination of grapes and grape products with ochratoxin A (OTA). The combined effect of temperature and RH on conidial germination of A. carbonarius on SGM and grape skin was described by combining Beta and polynomial equations. The equations developed in this work provided a good fit of the biological processes; they could be integrated in a predictive model for infection and OTA prediction in ripening grapes.     

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.     

Enhancement of the biocontrol agent Candida oleophila (strain O) survival and control efficiency under extreme conditions of water activity and relative humidity

The objective of this work is to evaluate the ability of some additive substances in protecting the biocontrol agent Candida oleophila (strain O) against the adverse effects of environmental factors, such as water activity (a_w, 0.93 and 0.98) and relative humidity (75% and 98%). The protection obtained with various protectant substances, skimmed milk (SM), peptone, maltose, sucrose, sorbitol, lactose and polyethylene glycol was assayed under in vitro and in vivo conditions. The yeast cells with the highest level of protecting agents (1%) had higher viability than those with low protectant levels (0.1% and 0.5%). SM, sucrose and sorbitol improved significantly the C. oleophila survival on apple fruit surface by 80.8%, 42.26% and 37.27% and gave a significant protection (from 96% to 100%) against Penicillium expansum under dried conditions. The highest strain O density and efficacy was obtained with SM. Under experimental conditions reflecting practical conditions, SM applied in combination with the strain O resulted in improved biocontrol efficacy by 74.65%. Therefore, SM could be used as material substrate with the best sugar protectants during the formulation process of this antagonistic yeast for eventual pre-harvest application.     

Compensatory growth responses to clipping defoliation in Leymus chinensis (Poaceae) under nutrient addition and water deficiency conditions

Compensatory growth responses of Leymus chinensis, a dominant species in Inner Mongolia steppe, to clipping defoliation were evaluated in a pot-cultivated experiment under different nutrient (N and P) and water availability conditions. Leymus chinensis exhibited over-compensatory growth at the light and moderate clipping intensities (20% and 40% aerial mass removed) with a greater accumulated aboveground biomass, higher relative growth rate (RGR), more rhizomatic tillers and a stimulation of compensatory photosynthesis to the remnant leaves as compared with those of the unclipped plants. Intense clipping (80% aerial mass removed), which removed most of the aboveground tissues, greatly reduced the growth of aboveground biomass in comparison with that of the unclipped plants. Nitrogen addition only slightly improved the biomass production and RGR in light and moderately clipped plants, and it did not allow plants in the intense clipping condition to over-compensate. Phosphorus addition had no obvious influences on the growth and physiological responses to clipping defoliation. These results indicated that nutrient addition could not compensate for the negative effects of severe clipping on the defoliated grass. On the other hand, there were no distinct positive responses under water deficiency condition for L. chinensis at all clipping intensities with a significant reduction of aboveground and belowground biomass, lower RGR, fewer rhizomatic tillers, and a lower net photosynthetic rate than other wet treatments. Additionally, the chlorophyll contents of remnant leaves gradually increased with the increase of clipping intensities in each treatment. In conclusion, although L. chinensis could compensate for tissues removal by some morphological and physiological responses, intense clipping and drought can result in a significant decrease of biomass and growth rate, even under enriched nutrition conditions.     

Response to joint selection on germination and flowering phenology depends on the direction of selection

Flowering and germination time are components of phenology, a complex phenotype that incorporates a number of traits. In natural populations, selection is likely to occur on multiple components of phenology at once. However, we have little knowledge of how joint selection on several phenological traits influences evolutionary response. We conducted one generation of artificial selection for all combinations of early and late germination and flowering on replicated lines within two independent base populations in the herb Campanula americana. We then measured response to selection and realized heritability for each trait. Response to selection and heritability were greater for flowering time than germination time, indicating greater evolutionary potential of this trait. Selection for earlier phenology, both flowering and germination, did not depend on the direction of selection on the other trait, whereas response to selection to delay germination and flowering was greater when selection on the other trait was in the opposite direction (e.g., early germination and late flowering), indicating a negative genetic correlation between the traits. Therefore, the extent to which correlations shaped response to selection depended on the direction of selection. Furthermore, the genetic correlation between timing of germination and flowering varies across the trait distributions. The negative correlation between germination and flowering time found when selecting for delayed phenology follows theoretical predictions of constraint for traits that jointly determine life history schedule. In contrast, the lack of constraint found when selecting for an accelerated phenology suggests a reduction of the covariance due to strong selection favoring earlier flowering and a shorter life cycle. This genetic architecture, in turn, will facilitate further evolution of the early phenology often favored in warm climates.     

Sample size and water dynamics on germinating diaspores: the first step for physiological and molecular studies on the germination process

Water dynamics on germinating diaspores is normally used as a chronological marker in molecular biology and physiological studies focusing on germination. However, there is no protocol to analyze this process, which may undermine all results generated. In this context, we determined the sufficient sample size to study water dynamics on germinating diaspores by means of a modified maximum curvature method (MMCM). As a biological model, we used diaspores from agricultural species with different physiological qualities. These diaspores were put in contact with distilled water and were analyzed regarding mass, every hour, until embryo protrusion. Data collected in the last observation were used to measure the sufficient sample size. Here, we show that (i) MMCM is an adequate method to measure the variability of water dynamics on germinating diaspores; (ii) the b coefficient can be used to infer the homogeneity of water dynamics on diaspores of different species as a function of the increase in sample size; (iii) in general, to study this process in an easier, safer and standardized way, seven diaspores are needed and (iv) water dynamics on germinating diaspores estimated by weighted mass demonstrated to be a very stable process which depends more on the species than on the physiological quality. We also discussed why the sample size and water dynamics on germinating diaspores should be the first step for protocols on physiological and molecular aspects of the germination process.     

The effect of plant growth regulators, nitric oxide, nitrate, nitrite and light on the germination of dimorphic seeds of Suaeda salsa under saline conditions

Suaeda salsa, a leaf succulent shrub in the family Chenopodiaceae, is one of the most important halophytes in China. Suaeda salsa produces dimorphic seeds (soft brown seeds and hard black seeds). Seeds of S. salsa were collected from the coastal salt flats near Huanghua City, China. Experiments were conducted to determine the salinity-alleviating effect of plant growth regulators, nitric oxide, nitrate, nitrite and light on the germination of dimorphic seeds of S. salsa. Brown seeds had a higher germination rate than black seeds in all experiments. Black seeds were more sensitive to salt in the absence of light in comparison to brown seeds. Brown seeds absorbed water more quickly in comparison to black seeds and were found to be more tolerant of salt stress. Our results showed that 1-aminocyclopropane-1-carboxylate (ACC, the immediate precursor of ethylene), nitrite, GA₄ and BA improved seed germination in the presence of salt. However, nitrate, GA₁, GA₃ failed to alleviate salt stress. ABA inhibited seed germination and seedling growth. Possible mechanisms involved in the alleviation of salt stress in S. salsa seeds and the ecological adaptation of the seeds to the environment are discussed.     

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.     

Storage behavior of Typha latifolia pollen at low water contents: Interpretation on the basis of water activity and glass concepts

Germplasm must be stored under optimal conditions to maximize longevity and efficiently maintain genetic resources. In order to identify optimal storage conditions, we investigated the effects of temperature (−5 to 45°C) and water content (<0.17 g H₂O g⁻¹ dry weight) on longevity of Typha latifolia L. pollen. Longevity was highest at water contents corresponding to storage relative humidity (RH) of 11‐15% which corresponded to the shoulder of water sorption isotherms. Also coinciding with this shoulder were abrupt changes in heat capacity of water present in the pollen. Consistent with changes in isotherms with temperature and the concept of critical RH for storage, optimum water contents increased with decreasing temperature. An attempt was made to explain the aging behavior according to the glass concept. The water content‐temperature combinations of optimal storage were found to be below the glass transition curve, indicating that optimum storage conditions are achieved when intracellular glasses are present. We also found a change in activation energy of aging in Arrhenius plots around T_g, demonstrating a change in aging kinetics when the glassy state is lost. We concluL that T_g curves cannot be used solely to predict precise conditions of optimum storage, but might be useful for predictions of storage longevity above optimum water contents. The data imply that too much drying reduces longevity and should be avoided, particularly when cryogenic storage is considered.     

Community-wide germination strategies in an alpine meadow on the eastern Qinghai-Tibet plateau: phylogenetic and life-history correlates

In this study, we built up a database of 633 species (48 families, 205 genera) from an alpine meadow on the eastern Qinghai-Tibet plateau. Our objective was to assess the effects of phylogenetic and life-history (life form, perenniality, seed size, dispersal strategy and period) background on the community-wide germination strategies. We found that the seeds of shrubs, perennials, and well-dispersed plants, and the smaller seeds germinated more and comparatively earlier. In one-way ANOVAs, phylogenetic groups explained 12% of the variance in GT (mean germination time for all seeds germinated of each species); life-history attributes, such as seed size, dispersal strategy, perenniality and life form explained 10%, 7%, 5%, and 1% respectively, and dispersal period had no significant effect on GT. Multifactorial ANOVAs revealed that the three major factors contributing to differences in GT were phylogenetic relatedness, seed size and dispersal strategy (explained 4%, 5% and 4% of the interspecific variation independently, respectively). Thus, seeds germination strategies were significantly correlated with phylogenetic and life-history relatedness. In addition, phylogenetic relatedness had close associations and interactions with seed size and dispersal strategy. Then, we think phylogeny and life-history attributes could not be considered mutual exclusively. Seed germination, like any other trait, is shaped by the natural history of the species and by the evolutionary history of the lineage. And a large percentage of the variance remained unexplained by our model, which suggested important selective factors or parameters may have been left out of this analysis. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Influence of temperature and water vapour pressure on the flight activity ofSimulium arcticum Malloch (Diptera: Simuliidae)

The effects of temperature and vapour pressure deficit on the flight activity ofSimulium arcticum were defined under controlled laboratory conditions. Optimum conditions of temperature and vapour pressure deficit for flight activity varied with time of exposure to the environmental conditions. By applying polynomial equations for the various response surfaces it was possible to predict the time-dependent, optimal hygrothermal environment for black-fly flight activity. Greatest variability in flight activity occurred just prior to death of the insects. Mortality of flies after 1 h exposures to the experimental conditions occurred only at high temperatures and vapour pressure deficits. With increased time of exposure, mortality increased at lower temperatures and vapour pressure deficits.     

Influence of in vitro growth conditions on photosynthetic competence and survival rate of Rehmannia glutinosa plantlets during acclimatization period

The photosynthetic responses of Rehmannia glutinosa grown under photoautotrophic or heterotrophic conditions in vitro were investigated after transfer to greenhouse conditions. In addition, the changes in carbohydrate content and survival rates of the plantlets were evaluated. During six days after transplantation, the photosynthetic rate declined and photoinhibitory impairments represented by decrease of Fv/Fm and chlorophyll content were observed regardless of environmental conditions in vitro. Excessive transpiration was observed in plantlets grown under heterotrophic conditions during that period. Fructose and glucose content of the plantlets grown under photoautotrophic conditions increased with time and reached almost the same level of field grown plants after day 15. Under heterotrophic conditions, in contrast, the content of these sugars decreased continuously during that period. It is suggested that high survival rate of plantlets grown under photoautotrophic conditions has to be attributed to improvement of photosynthetic competence by imposed high light intensity and CO₂ concentration in vitro. The results strongly suggest that the control of transpiration during early stage after transplantation plays a key role in the acclimatization process, and photoautotrophic conditions could be a solution to solve the problems associated with transplantation stress. This revised version was published online in June 2006 with corrections to the Cover Date.     

Interaction between elevated atmospheric concentration of CO2 and humidity on plant growth: comparison between cotton and radish

Cotton plants (Gossypium hirsutum L. var Deltapine 90) and radish plants (Raphanus sativus L var Round Red) were grown under full sunlight using a factorial combination of atmospheric CO₂ concentrations (350 µmol mol^-1 and 700 µmol mol^-1) and humidities (35% and 90% RH at 32 °C during the day). Cotton plants showed large responses to increased humidity and to doubled CO₂. In cotton plants, the enhanced dry matter yield due to doubled CO₂ concentration was 1.6-fold greater at low humidity than at high humidity. Apart from the direct effect of elevated CO₂ level on photosynthesis, the greater effect of doubled CO₂ concentration on dry matter yield at low humidity was probably due to: (1) increased leaf water potential caused by reduction of transpiration resulting from the negative CO₂ response of stomata to increased CO₂ concentration the consequence being greater leaf area expansion; (2) reduction of CO₂ assimilation rate at low humidity and normal CO₂ concentration as a result of humidity response of stomata causing reduction of intercellular CO₂ concentration. In contrast, apart from the very early stage of development, radish plants do not respond to increased humidity but had a relatively large response to doubled CO₂ concentration. Furthermore, due to the determinate growth pattern as well as having a prominent storage root, the extra photoassimilate derived at doubled CO₂ level is allocated to the storage root.Abbreviatios DAE day after emergence - LAD leaf areal density (leaf dry weight/leaf area) - LAR leaf area ratio (leaf area/plant dry weight) - NAR net assimilation rate - c_i internal CO₂ concentration - PPFD photosynthetic photon flux density - RGR relative growth rate - RLAGR relative leaf area growth rate - VPD vapour pressure deficit