Cross-resistance of the pathogenic fungus Alternaria alternata to fungicides with different modes of action.

BACKGROUND: Cross-resistance, a phenomenon that a pathogen resists to one antimicrobial compound also resists to one or several other compounds, is one of major threats to human health and sustainable food production. It usually occurs among antimicrobial compounds sharing the mode of action. In this study, we determined the sensitivity profiles of Alternaria alternata, a fungal pathogen which can cause diseases in many crops to two fungicides (mancozeb and difenoconazole) with different mode of action using a large number of isolates (234) collected from seven potato fields across China. RESULTS: We found that pathogens could also develop cross resistance to fungicides with different modes of action as indicated by a strong positive correlation between mancozeb and difenoconazole tolerances to A. alternata. We also found a positive association between mancozeb tolerance and aggressiveness of A. alternata, suggesting no fitness penalty of developing mancozeb resistance in the pathogen and hypothesize that mechanisms such as antimicrobial compound efflux and detoxification that limit intercellular accumulation of natural/synthetic chemicals in pathogens might account for the cross-resistance and the positive association between pathogen aggressiveness and mancozeb tolerance. CONCLUSIONS: The detection of cross-resistance among different classes of fungicides suggests that the mode of action alone may not be an adequate sole criterion to determine what components to use in the mixture and/or rotation of fungicides in agricultural and medical sects. Similarly, the observation of a positive association between the pathogen's aggressiveness and tolerance to mancozeb suggests that intensive application of site non-specific fungicides might simultaneously lead to reduced fungicide resistance and enhanced ability to cause diseases in pathogen populations, thereby posing a greater threat to agricultural production and human health. In this case, the use of evolutionary principles in closely monitoring populations and the use of appropriate fungicide applications are important for effective use of the fungicides and durable infectious disease management.

Trade-offs and evolution of thermal adaptation in the Irish potato famine pathogen Phytophthora infestans.

Temperature is one of the most important environmental parameters with crucial impacts on nearly all biological processes. Due to anthropogenic activity, average air temperatures are expected to increase by a few degrees in coming decades, accompanied by an increased occurrence of extreme temperature events. Such global trends are likely to have various major impacts on human society through their influence on natural ecosystems, food production and biotic interactions, including diseases. In this study, we used a combination of statistical genetics, experimental evolution and common garden experiments to investigate the evolutionary potential for thermal adaptation in the potato late blight pathogen, Phytophthora infestans, and infer its likely response to changing temperatures. We found a trade-off associated with thermal adaptation to heterogeneous environments in P. infestans, with the degree of the trade-off peaking approximately at the pathogen's optimum growth temperature. A genetic trade-off in thermal adaptation was also evidenced by the negative association between a strain's growth rate and its thermal range for growth, and warm climates selecting for a low pathogen growth rate. We also found a mirror effect of phenotypic plasticity and genetic adaptation on growth rate. At below the optimum, phenotypic plasticity enhances pathogen's growth rate but nature selects for slower growing genotypes when temperature increases. At above the optimum, phenotypic plasticity reduces pathogen's growth rate but natural selection favours for faster growing genotypes when temperature increases further. We conclude from these findings that the growth rate of P. infestans will only be marginally affected by global warming.

[Research on the Method of Improving Terahertz Frequency Resolution Based on Empirical Mode Decomposition].

Terahertz frequency resolution is an important factor affecting substance identification, but the presence of the device reflection within the terahertz time domain spectroscopy systems, causes the presence of a plurality of reflection peaks in the reference and measurement signals with low frequency resolution, because of the length truncation of time domain signal. In order to remove the influence of the reflection peak, this paper proposes a method based on empirical mode decomposition to remove the time domain reflection peak, to improve the terahertz frequency resolution. The time domain reflection peaks are positioned by correlation with the real terahertz peak, calculating the reflection peak upper and lower envelope and an average, obtaining intrinsic mode functions, and alternating reflection peaks with intrinsic mode functions, the effective length of the time domain signal is increased to improve the terahertz frequency resolution. Water vapor in the air terahertz transmission spectroscopy results show that this method can self adaptively remove a plurality of reflection peaks and has good repairing effect on the terahertz time-domain signal; the frequency resolution is increased by 12 times; and the useful information of absorption spectrum is not lost; the absorption peak position and the number is consistent with real spectrum; the terahertz spectrum ability to identify is well preserved.

[Absorption Characteristics and Simulation of LLM-105 in the Terahertz Range].

2,6-diamino-3,5-dinitropyrazine-1-oxide (LLM-105), a novel explosive with high energy and low sensibility. In order to study the molecular structure characteristics of the explosive, the absorption spectra of LLM-105 in the frequency range of 0.2-2.4 THz were detected by terahertz time-domain spectroscopy (THz-TDS). The results showed that a number of characteristic absorption peaks with different intensity located at 1.27, 1.59, 2.00, 2.08, 2.20, 2.29 THz. The article also simulated the absorption spectra of LLM-105 molecular crystal within 0.2-2.5 THz region by using Materials Studio 6.0 software based on density functional theory (DFT), and the simulated results agreed well with the experimental data except for the peak at 2.29 THz, which verified theoretically the accuracy of the experimental data. In addition, the vibrational modes of the characteristic peaks in the experimental absorption spectra were analyzed and identified, the results showed that the forming of the characteristic absorption peaks and the molecular vibration were closely related, which further provided important laboratory and technology support for the study of the transformation of molecule structure of LLM-105. There was no simulated frequency agreed with the experimental absorption peak at 2.29 THz, which may be caused by the vibration of the crystal lattice or other reasons.

[Experimental Conditions of Laser Induced Breakdown Spectroscopy of Metal Elements in Cement].

Laser induced breakdown spectroscopy (LIBS) is a widely used material element detection technology. Because of its detection result is affected by many factors, and therefore, analysing and comparising the different experimental conditions have important significance for LIBS. Experimental sample produced by Beichuan County, Sichuan Province, China, which is ordinary Portland cement P. O42.5, using eight-channel fiber optic spectrometer AvaSpec-2048-USB2-RM, delay trigger DG645 for LIBS testing. Several metallic elements as Mg, Al, Na, K, which affect cement's technical indicators were analyzed. Mainly compares the effect of laser frequency, the same point measurement times on different metal element spectral signal intensity, the optimum experimental parameters under the condition of this experiment: 10 Hz was the best laser frequency. When laser frequency is 10 Hz, the spectrum intensity of elements Mg, Al, Na, K were increased by 67.66%, 47.88%, 84.59%, 43.36% than 8 Hz. Because the tablet samples in place, the surface will have a small amount of oxidation and deliquescence, in order to measure 10 times for an average income results were recorded under the condition, with third, four records of results for the best.

[Acidity and temperature effect on the fluorescence characteristics of hydraulic oils and lubricants].

By analyzing HyJet V phosphate ester hydraulic oil environmental impacts (oil, etc.) and confounding factors (pH, temperature, etc.), the feasibility was studied for the fluorescence detection of aircraft hydraulic oil leaks. By using the fluorescence spectrophotometer at various acidities and temperatures, the fluorescence properties of HyJet V phosphate ester hydraulic oil, Jet Oil II lubricant and 2197 lubricant were gained. The experimental results are shown as following: The fluorescence peaks of HyJet V phosphate ester hydraulic oil, Jet Oil II lubricant and 2197 lubricant are at 362, 405 and 456 nm, respectively. The impact of temperature on HyJet V phosphate ester hydraulic oil is less effective; Jet Oil II lubricant and 2197 lubricant fluorescence intensity decreases with increasing temperature. When acidity increases, the fluorescence peak of HyJet V phosphate ester hydraulic oil gradient shifts from 370 to 362 nm, and the fluorescence intensity decreases; the fluorescence peak of Jet Oil II lubricant is always 405 nm, while the fluorescence intensity decreases; the fluorescence peak of 2197 lubricant at 456 nm red shifts to 523 nm, and double fluorescence peaks appeare. The results are shown as following: under the influence of the environment and interference factors, the fluorescence characteristics of HyJet V phosphate ester hydraulic oil remain unchanged, and distinguish from Jet Oil II lubricant and 2197 lubricant. Therefore, the experiments indicate that the detection of HyJet V phosphate ester hydraulic oil leak is feasible by using fluorescence method.

Low-dose decitabine for refractory thrombocytopenia following allogeneic hematopoietic stem cell transplantation in children: A pilot study.

Refractory thrombocytopenia is a critical complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT), which is not sensitive to conventional treatment and often leads to lower overall survival and disease-free survival. Previous studies have showed the efficacy and safety of low-dose decitabine for adults' refractory prolonged isolated thrombocytopenia in hematologic malignancy after allo-HSCT. However, clinical data on pediatric patients or non-hematologic malignancies are lacking. Herein, we evaluated the safety and efficacy of low-dose decitabine in nine children with persistent thrombocytopenia after HSCT. Patients received decitabine at 3.5 mg/m2, 5 mg/m2 or 10 mg/m2 respectively for three to five consecutive days according to underlying diseases and hyperplastic state of bone marrow. Six patients reached sustained platelets count more than 100 × 109/L, two patients achieved platelet transfusion independence. The total response rate was 88.8 % (8/9). One patient died from severe infection because of persistent agranulocytosis longer than 3 weeks. In conclusion, the present study supports the safety and efficacy of low-dose decitabine for treatment of refractory thrombocytopenia after allogeneic HSCT in children.

[Effect of temperature and pH on the fluorescence characteristic of oily waste water].

The present study was about the effect of temperature (5-55 degrees C) and pH (3-12) value on the fluorescence characteristic of oily waste water from the export of China Petroleum & Chemical Oil Refinery. It was found that temperature only affects the fluorescence intensity but not the site of fluorescence peaks. The fluorescence peaks shift right that from 413 to 426.5 nm when the oily waste water is acid. And along with the enhancing of the acidity the fluorescence intensity ascended. The fluorescence intensity decreased with alkaline enhancing but the site of fluorescence peaks remained unchanged at 398 nm. Temperature and pH value were the two important impact factors on the fluorescence characteristic of oily waste water. If desired it needs correction and compensation on temperature and pH value. That's expected to improve the detection accuracy of oil class and content.

[Feasibility of using laser-induced fluorescence to detect directly polycyclic aromatic hydrocarbons in soil].

Abstract In the present paper, a technique of laser-induced fluorescence(LIF)for direct assay of polycyclic aromatic hydrocarbons(PAH) in soil was put forward. The research objective of this article is anthracene. The possibility of using LIF spectra to detect directly anthracene in soil was studied. Anthracene was detected in soil by AvaSpec-3648 Fiber Optic Spectrometer of thermoelectric refrigeration. The authors drew a conclusion that in the range of certain anthracene concentration(0.000 005-0.001 g x g(-1)), the intensity of LIF fluorescence is linear with anthracene concentration in soil, with a regression coefficient of 0. 929. This showed that direct assay of anthracene in soil was feasible by laser-induced fluorescence. The study is important to developing a new analytical technique of quantitative fluorescence detector which can be applied to the analysis of PAH in soil without pretreatment, and is significant to realization of real-time, in-line, in-situ measurement of PAH in soil.

Increasing temperature elevates the variation and spatial differentiation of pesticide tolerance in a plant pathogen.

Climate change and pesticide resistance are two of the most imminent challenges human society is facing today. Knowledge of how the evolution of pesticide resistance may be affected by climate change such as increasing air temperature on the planet is important for agricultural production and ecological sustainability in the future but is lack in scientific literatures reported from empirical research. Here, we used the azoxystrobin-Phytophthora infestans interaction in agricultural systems to investigate the contributions of environmental temperature to the evolution of pesticide resistance and infer the impacts of global warming on pesticide efficacy and future agricultural production and ecological sustainability. We achieved this by comparing azoxystrobin sensitivity of 180 P. infestans isolates sampled from nine geographic locations in China under five temperature schemes ranging from 13 to 25°C. We found that local air temperature contributed greatly to the difference of azoxystrobin tolerance among geographic populations of the pathogen. Both among-population and within-population variations in azoxystrobin tolerance increased as experimental temperatures increased. We also found that isolates with higher azoxystrobin tolerance adapted to a broader thermal niche. These results suggest that global warming may enhance the risk of developing pesticide resistance in plant pathogens and highlight the increased challenges of administering pesticides for effective management of plant diseases to support agricultural production and ecological sustainability under future thermal conditions.

[Study on the fluorescent characteristic of tryptophan in the process of the waste water treatment].

Tryptophan is one kind of representative pollutants in waste water. The present paper focuses on the research on the relation between the consistence, pH, ionic strength and the fluorescent characteristic of tryptophan. The result shows that: (1) Tryptophan in the low consistence range (0.01-3 m x L(-1)) shows good linear relation, the correlation coefficient is up to 0.995 88, while the linear relation in the high consistence range (3-30 mg x L(-1)) is slightly worse and the correlation coefficient is only 0.942 24; (2) In the acidic condition, the relative fluorescence intensity of tryptophan strengthens while the pH arises, and when the pH is in the range (6.5, 7.5), the intensity is relatively steady, while when the pH reaches 12, it drops off; (3) With the enhancement of the ionic strength, the relative fluorescence intensity of tryptophan reduces to some extent, and when the nitrate ion reaches to a high consistence, the spectrogram shows EERS.

[The preliminary research on information redundance in fluorescence spectral data process].

This paper presented the problem of information redundance factor in spectral data analysis. Three-component mixtures fluorescence spectra data of anthracene, pyrene and phenanthrene, which were typical polycyclic aromatic hydrocarbons (PAHs), were investigated through principal component regression analysis and move windows wavelength selection in chemometrics. Theoretical analysis and experimental result demonstrated that there was information redundancy in fluorescence signal; the data which have been optimized by redundancy techniques could show more realistic information of the test samples quantitatively, and for the multi-component mixed system with spectral overlapped seriously, analysis with the data which have been optimized through redundancy techniques could improve the sensitivity and stability of the models.

[The quantitative analysis of polycomponent PAHs by netural network based on data synthese and principal].

The present paper used synthesized data from the experiment samples to replace partial basic experiments, and increased the training samples amount from 14 to 27. In principal component analysis (PCA), the dimensionality of multivariate data was reduced to n principal components and almost all data information was kept. The PCA reduced the network's input nodes from 60 to 3 to simplify the neural network's structure. Finally, back-propagation neural network was used to train and predict these samples. It had 27 training samples, the input layer had three nodes, the hidden layer had two nodes, and the output layer had two nodes. Its excitation function is variable learning rate method. The results show that the coefficient of recovery can reach 89.6-109.0. It has reached the expected purpose.

Lack of gene flow between Phytophthora infestans populations of two neighboring countries with the largest potato production.

Gene flow is an important evolutionary force that enables adaptive responses of plant pathogens in response to changes in the environment and plant disease management strategies. In this study, we made a direct inference concerning gene flow in the Irish famine pathogen Phytophthora infestans between two of its hosts (potato and tomato) as well as between China and India. This was done by comparing sequence characteristics of the eukaryotic translation elongation factor 1 alpha (eEF-1α) gene, generated from 245 P. infestans isolates sampled from two countries and hosts. Consistent with previous results, we found that eEF-1α gene was highly conserved and point mutation was the only mechanism generating any sequence variation. Higher genetic variation was found in the eEF-1α sequences in the P. infestans populations sampled from tomato compared to those sampled from potato. We also found the P. infestans population from India displayed a higher genetic variation in the eEF-1α sequences compared to China. No gene flow was detected between the pathogen populations from the two countries, which is possibly attributed to the geographic barrier caused by Himalaya Plateau and the minimum cross-border trade of potato and tomato products. The implications of these results for a sustainable management of late blight diseases are discussed.

[Comparison between PARAFAC and SWATLD based on four components simultaneous determination].

Catechol and hydroquinone are toxic substances, while tyrosine and tryptophan are luminescent substances in natural amino acids. Because of spectra overlap of the four components, it is difficult to detect quantity directly with conventional methods. A simple and reproducible method which provides simultaneous determination of catechol, hydroquinone, tyrosine and tryptophan in the same samples is presented in the present paper. The method combined excitation-emission fluorescence spectra with PARAFAC and SWATLD. At the same time, the differences between the two methods in qualitative determination and quantitative determination are compared. When PARAFAC was applied and the component number was set to 4, the obtained recoveries of hydroquinone, catechol, tyrosine and tryptophan were (101.2 +/- 2.7)%, (99.3 +/- 3.0)%, (98.7 +/- 4.5)% and (101.6 +/- 4.7)%, respectively. Meanwhile, the recoveries of the four components were (109.0 +/- 8.0)%, (91.3 +/- 11)%, (99.7 +/- 13)% and (98.1 +/- 11)% respectively with SWATLD method. The satisfactory results showed that the two methods can be used to determinate solution containing four components directly. By comparing the algorithms, PARAFAC algorithm was found to be more advantageous than SWATLD algorithm.

[Extraction of characteristic parameters of three-dimensional fluorescence spectra of tyrosine and tryptophan].

In the present paper, three-dimensional fluorescence spectra of tyrosine and tryptophan were detected through Pekin-Elmer LS55 fluorescence spectrometer made in U. S. A, with the excitation wavelength in the range of 230-320 nm, interval of 5 nm, emission wavelength in the range of 230-500 nm, and interval of 2 nm. Three-dimensional fluorogram was obtained using excitation wavelength, emission wavelength and fluorescence intensity detected by Pekin-Elmer LS55 as threedimensional system of coordinate. It can be seen that the main peak of the three-dimensional fluorescence spectra of tyrosine and tryptophan was aliasing evidently, so it was impossible to distinguish these two overlapping components using excitation wavelength, emission wavelength and fluorescence intensity easily. Therefore, to distinguish spectra of tyrosine and tryptophan obviously, characteristic parameter was extracted based on the principle of mathematical statistics and the most relevant information of the fluorescence spectra of tyrosine and tryptophan was obtained. The results showed that the difference value percentage of the "mean", "deviation", "origin pitch" and "mixed center pitch" of the three-dimensional spectra' s characteristic parameter was 330.37%, 102.86%, 329.16% and 329.63%, respectively; meanwhile, the difference value percentage of "distribution" and "correlation" was 10.61% and 2.40% respectively. Thus, it was obvious that the "mean", "deviation", "origin pitch" and "mixed center pitch" of the three-dimensional spectra's characteristic parameter can be used to distinguish overlap spectra of tyrosine and tryptophan as sensitive characteristic parameters. The principle and results of this method are applicable and valid. This "math pre-extracted" three-dimensional spectrometry found sensitive parameter among the components by the parameter extracting and can replace traditional excitation-emission-intensity three-dimensional fluorescence analysis, then can be generalized to the identification of multi-components.

Strategies of Phytophthora infestans adaptation to local UV radiation conditions.

Expected global changes in environmental conditions underline the need for a better understanding of genetic variation in ecological traits and their strategies of adaptation to the stresses. In this study, evolutionary mechanisms and processes of UV adaptation in plant pathogens were investigated by combining statistical genetics, physiological assays, and common garden experiment approaches in an assessment of the potato late blight pathogen, Phytophthora infestans, sampled from various geographic locations in China. We found spatial divergence caused by diversifying selection in UV tolerance among P. infestans populations. Local UV radiation was the driving force of selection as indicated by a positive correlation between UV tolerance in P. infestans populations and the altitude of collection sites. Plasticity accounted for 68% of population variation while heritability was negligible, suggesting temporary changes in gene expression and/or enzymatic activity play a more important role than permanent modification of gene structure in the evolution of UV adaptation. This adaptation strategy may explain the lack of fitness penalty observed in genotypes with higher UV tolerance.

Constraining Evolution of Alternaria alternata Resistance to a Demethylation Inhibitor (DMI) Fungicide Difenoconazole.

Evolution of fungicide resistance in plant pathogens is one of major concerns in sustainable plant disease management. In this study, the genetics and potential of developing resistance to a demethylation inhibitor (DMI) fungicide, difenoconazole, in the fungal pathogen Alternaria alternata was investigated using a comparative analysis of genetic variation in molecular (Single Sequence Repeats, SSR) and phenotypic (fungicide tolerance) markers. No difenoconazole resistance was found in the 215 A. alternata isolates sampled from seven different ecological zones in China despite the widespread use of the fungicide for more than 20 years. This result suggests that the risk of developing resistance to difenoconazole in A. alternata is low and we hypothesize that the low risk is likely caused by fitness penalties incurred by resistant mutants and the multiple mechanisms involving in developing resistance. Heritability and plasticity account for ∼24 and 3% of phenotypic variation, respectively, indicating that genetic adaptation by sequence variation plays a more important role in the evolution of difenoconazole resistance than physiological adaptation by altering gene expression. Constraining selection in the evolution of A. alternata resistance to difenoconazole was documented by different patterns of population differentiation and isolate-by-distance between SSR markers and difenoconazole tolerance. Though the risk of developing resistance is low, the findings of significant differences in difenoconazole tolerance among isolates and populations, and a skewing distribution toward higher tolerance suggests that a stepwise accumulation of tolerance to the fungicide might be occurring in the pathogen populations. As a consequence, dynamic management programs guided by evolutionary principles such as spatiotemporal rotations of fungicides with different modes of action are critical to prevent the continued accumulation of tolerance or the evolution of resistance to difenoconazole and other DMI fungicides.

Human-Mediated Gene Flow Contributes to Metapopulation Genetic Structure of the Pathogenic Fungus Alternaria alternata from Potato.

Metapopulation structure generated by recurrent extinctions and recolonizations plays an important role in the evolution of species but is rarely considered in agricultural systems. In this study, generation and mechanism of metapopulation structure were investigated by microsatellite assaying 725 isolates of Alternaria alternata sampled from potato hosts at 16 locations across China. We found a single major cluster, no isolate-geography associations and no bottlenecks in the A. alternata isolates, suggesting a metapopulation genetic structure of the pathogen. We also found weak isolation-by-distance, lower among than within cropping region population differentiation, concordant moving directions of potato products and net gene flow and the highest gene diversity in the region with the most potato imports. These results indicate that in addition to natural dispersal, human-mediated gene flow also contributes to the generation and dynamics of the metapopulation genetic structure of A. alternata in China. Metapopulation structure increases the adaptive capacity of the plant pathogen as a result of enhanced genetic variation and reduced population fragmentation. Consequently, rigid quarantine regulations may be required to reduce population connectivity and the evolutionary potential of A. alternata and other pathogens with a similar population dynamics for a sustainable plant disease management.

Evidence for intragenic recombination and selective sweep in an effector gene of Phytophthora infestans.

Effectors, a group of small proteins secreted by pathogens, play a critical role in the antagonistic interaction between plant hosts and pathogens through their dual functions in regulating host immune systems and pathogen infection capability. In this study, evolution in effector genes was investigated through population genetic analysis of Avr3a sequences generated from 96 Phytophthora infestans isolates collected from six locations representing a range of thermal variation and cropping systems in China. We found high genetic variation in the Avr3a gene resulting from diverse mechanisms extending beyond point mutations, frameshift, and defeated start and stop codons to intragenic recombination. A total of 51 nucleotide haplotypes encoding 38 amino acid isoforms were detected in the 96 full sequences with nucleotide diversity in the pathogen populations ranging from 0.007 to 0.023 (mean = 0.017). Although haplotype and nucleotide diversity were high, the effector gene was dominated by only three haplotypes. Evidence for a selective sweep was provided by (i) the population genetic differentiation (GST) of haplotypes being lower than the population differentiation (FST) of SSR marker loci; and (ii) negative values of Tajima's D and Fu's FS. Annual mean temperature in the collection sites was negatively correlated with the frequency of the virulent form (Avr3aEM), indicating Avr3a may be regulated by temperature. These results suggest that elevated air temperature due to global warming may hamper the development of pathogenicity traits in P. infestans and further study under confined thermal regimes may be required to confirm the hypothesis.