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.     

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.     

桃褐腐病菌拮抗性内生细菌的筛选及其抑病效果

【目的】从桃树根部组织中分离、筛选能够防治桃褐腐病害的内生拮抗菌,从细胞学水平探究其抑制机理。【方法】采用平板对峙法筛选对桃褐腐病菌(Monilinia fructicola)有拮抗作用的内生细菌,调查内生拮抗细菌的形态学及生理生化特性,通过细菌16S r RNA基因测序分析菌株的系统发育学特征。通过果实离体实验检测内生拮抗细菌的抗菌效果,使用电子显微镜观察受抑制的桃褐腐病菌菌丝、孢子的形态以及细胞内部结构变化。【结果】从桃树根部组织中复筛分离得到的3株内生细菌对桃褐腐病防效高且稳定,经鉴定3株细菌均为枯草芽孢杆菌。果实离体试验表明,3株内生细菌均明显抑制了桃褐腐病的发生。通过电子显微镜观察,发现受拮抗细菌抑制的桃褐腐病菌的菌丝明显变细、杂乱且打结缠绕成团,孢子普遍干瘪变大、破裂,细胞质外渗。【结论】从桃树根部组织中得到的3株内生细菌对桃褐腐病菌的生长均有明显的抑制作用,可以作为防治桃褐腐病的新选择,具有进一步研究价值。     

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.     

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.     

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.     

离体条件下暴马丁香切开种子的萌发

分别以三种催芽处理10、20、40 d的暴马丁香种子进行离体培养试验,对种子做三种切割处理,中间切、两端切和完整种子。结果表明:采用切开种子的方法进行离体培养可提高种子发芽率,中间切种子的萌发最好,且在培养10 d后发芽率即达到最高值;经过40 d催芽处理的种子优于未经催芽和只进行20 d催芽处理的种子;切开种子以MS培养基附加5 mg/L BA或5 mg/LBA+0.1 mg/L IBA为最适培养基。     

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 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.     

Resilience of rice (Oryza spp.) pollen germination and tube growth to temperature stress

Resilience of rice cropping systems to potential global climate change will partly depend on the temperature tolerance of pollen germination (PG) and tube growth (PTG). Pollen germination of high temperature‐susceptible Oryza glaberrima Steud. (cv. CG14) and Oryza sativa L. ssp. indica (cv. IR64) and high temperature‐tolerant O. sativa ssp. aus (cv. N22), was assessed on a 5.6–45.4 °C temperature gradient system. Mean maximum PG was 85% at 27 °C with 1488 μm PTG at 25 °C. The hypothesis that in each pollen grain, the minimum temperature requirements (T_n) and maximum temperature limits (T_x) for germination operate independently was accepted by comparing multiplicative and subtractive probability models. The maximum temperature limit for PG in 50% of grains (T_x(50)) was the lowest (29.8 °C) in IR64 compared with CG14 (34.3 °C) and N22 (35.6 °C). Standard deviation (s_x) of T_x was also low in IR64 (2.3 °C) suggesting that the mechanism of IR64's susceptibility to high temperatures may relate to PG. Optimum germination temperatures and thermal times for 1 mm PTG were not linked to tolerating high temperatures at anthesis. However, the parameters T_x(50) and s_x in the germination model define new pragmatic criteria for successful and resilient PG, preferable to the more traditional cardinal (maximum and minimum) temperatures.     

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     

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。     

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.     

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.     

Effects of light, temperature and water stress on germination of Artemisia sphaerocephala

Artemisia sphaerocephala is widely used for vegetation rehabilitation, but its germination is very low after air seeding of achenes. We explored effects of light, temperature and water stress on germination. Results show that both final percent germination and germination rate were increased by temperature increment, with the highest values occurring at 15: 25°C (night: day) in dark and 20: 30°C under light. Light inhibited germination, especially at lower alternating temperatures (5: 15°C and 10: 20°C). The alternating temperature window for germination was slightly narrower under light than in dark, and germination was slower under light than in dark across the temperature range. Achenes incubated in the dark and at constant temperatures had over 80% germination at 10 to 25°C, with an optimum at 20°C. Under dark and 25μmol m^‐2 s^‐1 light flux density at 10: 20°C, final percent germination was over 94%, but if the light flux density was increased to 100 and 400 μmol m^‐2 s^‐1, final percent germination was significantly lower (64% and 38% respectively). However, achenes could keep their germination capacity for a long time (over 50 days) and germinate well after going back to the dark. Germination was also lower under water stress and few achenes germinated at ‐1.4 MPa. This was more pronounced at high and low temperatures. Given these findings and the prevailing climatic characteristics, the most suitable time for air seeding of achenes may be mid‐May.     

Local adaptation to different phytogeographic regions: habitat‐related variations in seed germination in response to temperature and salinity for two medicinal Salvia species from Jordan

Salvia spinosa L. and Salvia syriaca L. are perennial medicinal herbs that occur in the Mediterranean, Irano‐Turanian and Saharo‐Arabian phytogeographic regions of Jordan. With respect to the seed germination requirements, prevailing environmental conditions in each phytogeographic region may promote local adaptation and consequently affect the distribution range of the species. Using seeds of both species collected from populations across the three regions, we tested responses to variations in temperature and salinity under laboratory conditions. Both species showed significant differences in cumulative germination percentages and germination rates (modified Timson Index) with temperature, while origin only significantly affected S. spinosa seeds. Both species germinated best under the highest temperature regime (32/20°C). The low temperature regime (8/4°C) completely inhibited germination in S. syriaca, wheras it led to 80% to 95% germination in S. spinosa, with significant variation being recorded between the phytogeographic regions. For both species, salt solutions of 0, 25 and 50 mM NaCl yielded the highest germination percentages and rates, which sharply and significantly declined at higher concentrations (100 and 200 mM NaCl). Our results provide evidence of local adaptation of the study species to salinity and temperature in the respective maternal environments, particularly in the Irano‐Turanian and Saharo‐Arabian regions. Such differentiation should be accounted for in future conservation planning.     

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.