Two different PCR approaches for universal diagnosis of brown rot and identification of Monilinia spp. in stone fruit trees

AIMS: To design a protocol for the universal diagnosis of brown rot by polymerase chain reaction (PCR) in plant material and subsequently Monilinia spp. identification. METHODS AND RESULTS: Primers for discrimination of Monilinia spp. from other fungal genera by PCR were designed following a ribosomal DNA analysis. Discrimination among species of Monilinia was subsequently achieved by developing primers using SCAR (Sequence Characterised Amplified Region) markers obtained after a random amplified polymorphic DNA study. In addition, an internal control (IC) based on the utilization of a mimic plasmid was designed to be used in the diagnostic protocol of brown rot to recognize false negatives due to the inhibition of PCR. CONCLUSIONS: The four sets of primers designed allowed detection and discrimination of all Monilinia spp. causing brown rot in fruit trees. Addition of an IC in each PCR reaction performed increased the reliability of the diagnostic protocol. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection protocol presented here, that combined a set of universal primers and the inclusion of the plasmid pGMON as an IC for diagnosis of all Monilinia spp., and three sets of primers to discriminate the most important species of Monilinia, could be an useful and valuable tool for epidemiological studies. The method developed could be used in programmes to avoid the spread and introduction of this serious disease in new areas.     

Population dynamics of Epicoccum nigrum, a biocontrol agent against brown rot in stone fruit

Aims:  To study the population dynamics of Epicoccum nigrum on peaches and nectarines and to enhance its colonization on fruit surfaces to improve its biocontrol efficacy against brown rot.
Methods and Results:  Twelve surveys were performed to study E. nigrum populations and their effect on the number of the pathogenic Monilinia spp. conidia in peach orchards in Spain and Italy between 2002 and 2005. Fresh conidia and five different formulations of E. nigrum conidia were applied three to six times to peach and nectarine trees from full flowering to harvest. The size of the E. nigrum populations was determined from the number of colony-forming units and conidial numbers per flower or fruit. Treatment with all conidial formulations increased the size of the indigenous conidial population on peach surfaces.
Conclusions:  Formulations of E. nigrum having high viability are most effective against conidia of the pathogen when applied at pit hardening and during the month immediately before fruit harvest.
Significance and Impact of the Study:  Application of an E. nigrum conidial formulation decreased the number of conidia of Monilinia spp. on fruit surfaces during the growing season to the same extent as fungicides.     

Evaluation of commercial essential oil samples on the growth of postharvest pathogen Monilinia fructicola (G. Winter) Honey

Aim: To assess the effect of several commercial essential oils samples Australian lemon myrtle (Backhousia citriodora), cinnamon bark (Cinnamomum zeylanicum), oregano (Origanum vulgare), thyme oil (Thymus vulgaris), clove bud (Eugenia caryophyllata), valerian (Valeriana officinalis) and Australian tea tree oil (Melaleuca alternifolia) on mycelium growth and spore germination of Monilinia fructicola. The effectiveness of lemon myrtle essential oil as a fumigant for the control of brown rot in nectarines was evaluated. Methods and Results: Monilinia fructicola exhibited a different level of sensitivity to each tested essential oil with results suggesting that the essential oils provide excellent control of the pathogen with respect to mycelium growth and spore germination at very low concentrations, whereas for others higher concentrations are needed to reduce significant fungal growth. In vivo application of lemon myrtle essential oil effectively reduced the incidence of M. fructicola on noninoculated fruit. Fumigation of nectarines following inoculation did not reduce the incidence of brown rot in comparison with the inoculated control treatment. No evidence of phytotoxicity on the fruit was recorded. Conclusions: Lemon myrtle essential oil exhibited the strongest antifungal activity against M. fructicola, in vitro and to a lesser extent, under in vivo conditions. Significance and Impact of the Study: The results demonstrate that lemon myrtle essential oil, in particular, has potential as an antifungal agent to control M. fructicola.     

Antifungal effect of gaseous nitric oxide on mycelium growth, sporulation and spore germination of the postharvest horticulture pathogens, Aspergillus niger, Monilinia fructicola and Penicillium italicum

Aim:  To evaluate the antifungal activity of nitric oxide (NO) against the growth of the postharvest horticulture pathogens Aspergillus niger , Monilinia fructicola and Penicillium italicum under in vitro conditions.
Methods and Results:  Different volumes of NO gas were injected into the Petri dish headspace to obtain the desired concentrations of 50–500  μ l l⁻¹ . The growth of the fungi was measured for 8 days of incubation in air at 25°C . All concentrations of NO were found to produce an antifungal effect on spore germination, sporulation and mycelial growth of the three fungi, with the most effective concentration for A. niger and P. italicum being 100 and 500  μ l l⁻¹ for M. fructicola .
Conclusions:  Short-term exposure to a low concentration of NO gas was able to inhibit the subsequent growth of A. niger , M. fructicola and P. italicum .
Significance and Impact of the Study:  NO gas has potential use as a natural fungicide to inhibit microbial growth on postharvest fruit and vegetables.     

盐分和水分胁迫对盐生植物灰绿藜种子萌发的影响

研究了不同浓度的NaCl和复合盐及等渗溶液(PEG-6000)处理下盐生植物灰绿藜(Chenopodium glaucum L.)种子的萌发状况.结果表明:灰绿藜种子的萌发率与处理溶液的浓度或渗透势之间有显著的负相关关系;在低浓度盐溶液(2.9 g*L-1)中灰绿藜种子的萌发率高于对照(蒸馏水);NaCl溶液对灰绿藜种子萌发的抑制作用大于复合盐溶液.渗透势为-0.2和-0.5 mPa 时,PEG-6000溶液对灰绿藜种子萌发的抑制作用小于等渗NaCl溶液,而在较高渗透势溶液中则正好相反.用渗透势≤-1.8 mPa 的PEG-6000溶液及所有浓度的NaCl和复合盐溶液处理的种子复水后相对萌发率都达到了90%以上,说明一定程度的盐分和水分胁迫对灰绿藜种子萌发潜力并没有很大的影响,并且萌发恢复率随处理盐浓度或PEG-6000溶液渗透势(≤-1.4 mPa)的增加而增加.     

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

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

Effect of environmental factors on seed germination and emergence of Lepidium vesicarium

Lepidium vesicarium is a weed species with a wide distribution in the rangelands and dry‐land farming in East Azarbaijan, Iran. The experiments were undertaken to assay the effects of light, temperature, pH, osmotic potential, NaCl concentration and burial depth on seed germination and emergence of L. vesicarium. Germination was maintained at high levels (> 80%) over a wide day/night temperature range (10/5 to 30/20°C), but a severe reduction in the germination rate of L. vesicarium was found below 20/10°C. Germination of L. vesicarium was influenced by different light/dark regimes, as the germination rate was highest at 16 h light for the all treatments (0, 8, 12, 16 and 24 h light). Germination was 92–95% over a wide range of pH (2‐10). Germination was >50% at a water potential of ?0.7 MPa and salinity of 21 dS/m, indicating that drought and salt conditions have a minimal impact on seed germination. With increasing burial depth from 0 to 2 cm, the number of days required for 50% emergence increased and no germination was observed at burial depths deeper than 3 cm. This suggests that L. vesicarium would become troublesome in the rangelands and for growers in reduced‐tillage cropping systems. The ability to emerge from shallow depths, coupled with tolerance of a wide pH range, drought and salinity at germination, should be taken into account when managing this weed species.     

Effect of high temperature and water stress on pollen germination and spikelet fertility in rice

In future climates, rice could more frequently be subjected to simultaneous high temperature and water stress during sensitive developmental stages such as flowering. In this study, five rice genotypes were exposed to high temperature, water stress and combined high temperature and water stress during flowering to quantify their response through spikelet fertility. Microscopic analyses revealed significant differences in anther dehiscence between treatments and genotypes, with a moderately high association with the number of germinated pollen grains on the stigma. There was a strong relationship between spikelet fertility and the number of germinated pollen on stigmas. Although, all three stress treatments resulted in spikelet sterility, high-temperature stress caused the highest sterility in all five genotypes. A cumulative linear decline in spikelet fertility with increasing duration of independent high-temperature stress and in combination with water stress was quantified. Better anther dehiscence, higher in vivo pollen germination, and higher spikelet fertility were observed in both the N22 accessions compared with IR64, Apo and Moroberekan under high temperature, water stress and combined stress, indicating its ability to tolerate multiple abiotic stresses.     

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.     

Effect of light and temperature on the germination of lettuce seeds

A short period (15–30 min) at 30° C promotes germination of seeds of Lactuca sativa L. cv. Repolhuda in darkness. Far-red light reverses this stimulation, and the escape curves for phytochrome and high-temperature action are quite similar, indicating that the two factors act at a common point in the chain of events leading to germination. It is suggested that high temperature acts by decreasing the threshold of the active, far-red absorbing, form of phytochrome (Pfr) needed to promote germination.Abbreviations FR far-red light - Pfr far-red-absorbing form of phytochrome - R red light     

Effect of Radiation Spectral Composition on Nicotiana spp. Seedlings Grown in vitro

The aim of this work was to assess the responses of seedlings of five species of Nicotiana genus to red and far red radiation. N. acuminata exhibits positive photoblastic behaviour and germination was completely inhibited under far red and darkness. In N. glauca germination was reduced under far red and darkness, but the other species showed neutral behaviour. The hypocotyl elongation was inhibited in N. glauca and N. tabacum under white and far red radiation. In N. langsdorffii and N. debneyi hypocotyl was elongated under far red radiation. Only in N. acuminata red radiation promote greater hypocotyl elongation than dark condition. On the phylogenetic tree obtained from restriction analysis N. glauca and N. acuminata are grouped in one branch, while the other species, N. langsdorffii, N. debneyi and N. tabacum, are grouped in the other branch cluster. Moreover, the N. debneyi behaviours under different radiation treatments were similar to those of N. tabacum. These two species are allopolyploid members of the genus Nicotiana, as also was confirmed by this study. This revised version was published online in July 2006 with corrections to the Cover Date.     

光照和温度对百合属6种植物种子萌发的影响

对不同光照和温度条件对条叶百合（Lilium callosum Sieb.et Zucc.)、大花卷丹[L. leichtlinii Hook.f var.maximowicaii(Regel)Baker]、有斑百合[L.concolor Salisb.var.pulchellum(Fisch.)Regel]、川百合[L.davidii Duchartre)、毛百合（L. dauricum Ker-Gawl.)和东北百合（L.distichum Nakai)种子萌发的影响进行了研究。结果表明：光照对有斑百合、川百合和毛百合种子萌发有明显促进作用,可缩短种子萌发时间,提高种子萌发率。24h光照下种子萌发完全所需天数比12h光照少,种子萌发率以24h光照最佳。避光条件下温度对大花卷丹、有斑百合、毛百合、川百合及东北百合种子萌发率和萌发速度有影响,对条叶百合种子影响最大,其种子萌发最适温度为20℃,5～6d开始萌动,2～3周萌发完全,随着温度的升高或降低其种子萌发率下降。光照条件下,变温对种子萌发影响不明显。子叶留土类型的毛百合种子有二次休眼现象,9000lx光照能代替低温解除二次休眼。经不同前处理的百合种子萌发率和萌发速度不同。     

Effects of NaCl salinity in vivo and in vitro on ribonuclease activity of Vigna unguiculata cotyledons during germination

Pitiuba bean [ Vigna unguiculata (L.) Walp.] seeds were sown in water or. in 0.1 M NaCl. Seedling growth and cotyledon nucleic acid mobilization were delayed by NaCl salinity. The differences in cotyledonary RNase activity between seeds sown in water and in NaCl solutions suggest that salinity delays the activation and/or de novo synthesis of the enzyme. Cotyledon extracts were subjected to gel filtration through Sephadex G-100, and RNase activity measured. Only one cotyledonary RNase appeared during germination, and salinity did not induce any change in molecular weight of the enzyme. Salinity inhibited 45% of the specific activity of the RNase on the 5th day of the experimental period. The same salt concentration (0.1 M NaCl) added in vitro inhibited only 8 % of the specific activity of the enzyme. This difference may indicate that NaCl in vivo affects mainly the de novo synthesis of the RNase.     

Effect of temperature and water activity on mycelia radial growth,conidial production and germination of Lecanicillium spp. isolates and their virulence against Trialeurodes vaporariorum on tomato plants

Biocontrol of the whitefly Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) using entomopathogenic fungi has been a difficult challenge under greenhouse conditions. In order to select fungal isolates adapted to high temperature and extremely low moisture nine isolates of Lecanicillium lecanii (Zimmerman) Zare & W. Gams, L. attenuatum Zare & W. Gams and L. longisporum (Petch) Zare & W. Gams (Hypocreales: Clavicipitaceae) were evaluated. In vitro assays were performed to determine colony radial growth, conidial production and conidial germination in three water activity media (a_w = 0.97, 0.98 and 1.00) at 28 and 32 °C. Virulence of Lecanicillium spp. isolates was evaluated against third instar T. vaporariorum on tomato plants at 23 °C. Colony radial growth, conidial production and germination decreased with the reduction in water activity, while 32 °C was extremely detrimental for all fungal isolates. However, some isolates were able to grow and produce conidia at low water activity and high temperature. Additionally, mortality above 60 % was recorded for one of these isolates. Practical implementation of biocontrol of T. vaporariorum under greenhouse production systems should consider the selection of those Lecanicillium isolates that show tolerance to the adverse environmental conditions in greenhouses.     

Mathematical description of the seed germination dependency on time and temperature

Abstract. A mathematical model which describes the germination percentage dependency on time and temperature of a seed population was derived from the experimental results with a seed population of Amaranthus patulus Bertol. under sub-optimal temperature conditions (Washitani & Takenaka, 1984). The equation of the model which is a modified thermal time model is where G is germination percentage at a certain time after the start of imbibition ( t ) at a certain temperature ( T ), μ _{T 1} and σ _{T 1} are the mean and standard deviation of lower limit temperature among the seeds belonging to the seed population, and Tb, m , and A are the parameters characterizing the linear relationship between the rate and temperature, namely, Tb is the base temperature, m , the median of the required thermal time and A , a parameter determining the pattern of the variation of the required thermal time within seed population, respectively. The equation yields time courses for germination which are very similar to those observed by experiment.     

Interaction of light and temperature on seed germination of Rumex obtusifolius L.

Germination of Rumex obtusifolius L. seeds (nutlets) is low in darkness at 25° C. Germination is stimulated by exposure to 10 min red light (R) and also by a 10-min elevation of temperature to 35° C. A 10-min exposure to far-red light (FR) can reverse the effect of both R (indicating phytochrome control) and 35° C treatment. Fluence-response curves for this reversal of the effect of R and 35° C treatments are quantitatively identical. Treatment for 10 min with light of wavelenght 680, 700, 710 and 730 nm, after R and 35° C treatment, demonstrates that germination induced by 35° C treatment results from increased sensitivity to a pre-existing, active, far-red-absorbing form of phytochrome (Pfr) in the seeds.Abbreviations FR far-red light - P phytochrome - Pr red-absorbing form of P - Pfr far-red-absorbing form of P - R red light     

Seed biology and in vitro seed germination of Cypripedium

Abstract

Cypripedium orchids have high horticultural value. The populations of most species are very geographically restricted and they are becoming increasingly rare due to the destruction of native habitats and illegal collection. Reduction of the commercial value through large-scale propagation in vitro is a preferable option to reduce pressure from illegal collection. Cypripedium species are commercially propagated via seed germination in vitro. This review focuses on in vitro seed germination and provides an in-depth analysis of the seed biology of this genus.     

Effect of temperature and dark pretreatment on the germination of three species of Jamesonia (Pteridaceae,Polypodiopsida)

To study the influence of temperature on the germination ability of three species of Jamesonia (Jamesonia imbricata, Jamesonia scammaniae and Jamesonia rotundifolia), spores were cultured at 10°C, 15°C and 20°C. A temperature of 15°C was selected as representative of the natural annual average temperature of the paramo environment that Jamesonia species inhabit. In addition, a dark pretreatment of 2 days was tested to verify if germination was enhanced. The results indicated that germination of Jamesonia, considering the three species as a whole, is affected by temperature, but is independent of the dark treatment. All species showed higher and faster germination at 20°C, and exhibited a threshold minimum temperature around 10°C, below which germination is avoided or extremely low and delayed. This could suggest that spore germination in Jamesonia is adapted to establish gametophyte populations during frost‐free periods.     

Effect of temperature on the progamic phase in high-mountain plants

Progamic processes are particularly temperature-sensitive and, in lowland plants, are usually drastically reduced below 10 °C and above 30 °C. Little is known about how effectively sexual processes of mountain plants function under the large temperature fluctuations at higher altitudes. The present study examines duration and thermal thresholds for progamic processes in six common plant species (Cerastium uniflorum, Gentianella germanica, Ranunculus alpestris, R. glacialis, Saxifraga bryoides, S. caesia) from different altitudinal zones in the European Alps. Whole plants were collected from natural sites shortly before anthesis and kept in a climate chamber until further processing. Flowers with receptive stigmas were hand-pollinated with allopollen and exposed to controlled temperatures between -2 and 40 °C. Pollen performance (adhesion to the stigma, germination, tube growth, fertilisation) was quantitatively analysed, using the aniline blue fluorescence method. Pollen adhesion was possible from -2 to 40 °C. Pollen germination and tube growth occurred from around 0 to 35 °C in most species. Fertilisation was observed from 5 to 30-32 °C (0-35 °C in G. germanica). The progamic phase was shortest in G. germanica (2 h at 30 °C, 12 h at 5 °C, 24 h at 0 °C), followed by R. glacialis (first fertilisation after 2 h at 30 °C, 18 h at 5 °C). In the remaining species, first fertilisation usually occurred after 4-6 h at 30 °C and after 24-30 h at 5 °C. Thus, mountain plants show remarkably flexible pollen performance over a wide temperature range and a short progamic phase, which may be essential for successful reproduction in the stochastic high-mountain climate.     

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.