Fungicide Stress Research Articles

Similar recovery time of microbial functions from fungicide stress across biogeographical regions

Determining whether the structural and functional stress responses of communities are similar across space and time is paramount for forecasting and extrapolating the consequences of anthropogenic pressures on ecosystems and their services. Stream ecosystems are under high anthropogenic pressure; however, studies have only examined the response of stream communities across large scales over multiple generations. We studied the responses of leaf-associated microbial communities in streams within three European biogeographical regions to chemical stress in a microcosm experiment with multiple cycles of fungicide pollution and resource colonisation. Fungal community composition and the ecosystem function leaf decomposition were measured as response variables. Microbial leaf decomposition showed similar recovery times under environmental levels of fungicide exposure across regions. Initially, the decomposition declined (between 19 and 53%) under fungicide stress and recovered to control levels during the third cycle of pollution and colonisation. Although community composition and its stress response varied between regions, this suggests similar functional community adaptation towards fungicide stress over time. Genetic, epigenetic and physiological adaptations, as well as species turnover, may have contributed to community adaptation but further studies are required to determine if and to which extent these mechanisms are operating. Overall, our findings provide the first evidence of a similar functional response of microbial leaf decomposition to chemical stress across space and time.

Impact of benzimidazole and dithiocarbamate fungicides on the photosynthetic machinery, sugar content and various antioxidative enzymes in chickpea

Background and aimsFungicides, though beneficial for agricultural productivity, are known to interfere with the basic metabolism and induce the formation of various biomolecules and also alter the physiological parameters of plant growth. The present study is an attempt to understand the effect of different conc. of benzimidazole (Carbendazim) and dithocarbamate (Mancozeb) fungicides on photosynthetic components such as chlorophyll content, total sugar and phenolic content and various antioxidative enzymes in developing seedlings of chickpea. Material and Methods: Chickpea seeds of two cultivars (PDG-4 and GPF-2) were incubated with different conc. (0.1, 0.25 and 0.5%) of the fungicide for 24 and 48 h and then allowed to germinate for 10 days in an incubated chamber. Seedlings were analyzed for various physiological parameters such as variation in root/shoot length, photosynthetic activity (chlorophyll content), total sugar and phenolic content and activity of antioxidative enzymes such as GPX, CAT and SOD etc. Results and conclusionsCompared to the unstressed samples, fungicide stress resulted in an overall decrease in root/shoot length, relative water content etc. thus indicating that the applied fungicides adversely affects the rate of germination of seedlings. A differential behaviour of various chlorophyll (Chla, Chlb, total chlorophyll) contents suggests that fungicides stress affects the photosynthetic machinery. Estimations of sugar and total phenolic content indicated that higher conc. of the fungicide lowered the total sugar content at the 10-day-old seedling stage; thereby giving an indication that the fungicide may interferes with carbohydrate metabolism. We observed that the level of peroxidase increased at higher conc. of the both types of fungicide as compared to control samples whereas the catalase activity increased in PDG 4 but a lower activity was observed in GPF-2 under increasing conc. of both the fungicides. The levels of superoxide dismutase decreased in PDG-4 but increased in GPF-2 under higher conc. of both the fungicides thus indicating that different varieties of chickpea behaved differently and triggers various antioxidant enzymes as defence mechanism to counter the fungicides stress.

Initial state of soil microbial communities determines their stress response

The relationships between microbial communities biodiversity and soil functions are difficult to identify. In most cases, the establishment of these links requires alteration of microbial communities. In this work, we deliberately chose to subject soils to an unrealistic dose of biocide (fungicide 1000 times the recommended dose) in order to modify soil microbial communities. Two different soils were chosen from a luvisol, with contrasting land-uses: a long-term arable cropping plot (CC) and a permanent grassland (PG). Microbial communities were assessed by the measurement of the total microbial biomass via the quantification of total soil DNA. Total bacterial and fungal abundances and different bacterial taxa were measured by qPCR of rRNA genes. Results showed that fungicide stress significantly reduced total microbial and fungal biomass in PG and CC soils and decreased bacterial biomass only in CC soil. In both soils, fungicide stress decreased the abundance of Acidobacteria and Bacteroidetes. The Proteobacteria groups were insensitive or increased after fungicide addition. Enzyme activities and soil carbon mineralization were evaluated. In both soils, fungicide stress reduced cellulase, β-glucosidase and dehydrogenase activities, while N-acetyl-glucosaminidase was not affected. Xylanase activity was not affected in PG soil and it was inhibited in CC soil. Changes in structural and functional profiles of microbial communities were not correlated in the PG soil and were negatively correlated in the CC soil. To conclude, this work comfirms that the responses of microbial communities to stress depend on the nature of applied stress, measured functions and initial state of microbial communities.

Evidence for local adaptation and pleiotropic effects associated with melanization in a plant pathogenic fungus

We combined a common garden experimental design with digital image analysis to determine how melanization responds to temperature and fungicide stress in 126 strains of Rhynchosporium commune sampled from nine global field populations. We found that different temperatures and fungicide stress significantly affected the degree of melanization. The nine field populations showed similar patterns in response to the different temperatures. Significant correlations were found between the degree of melanization and the local environment, including mean annual temperature, latitude, and relative humidity, suggesting that melanization is a locally adaptive trait. We also found that melanization is positively correlated with both virulence and fungicide resistance. These findings suggest that melanization has pleiotropic effects in Rhynchosporium commune.

Novel gene-sequence markers for isolate tracking within Monilinia fructicola lesions.

Monilinia fructicola is a diverse pathogen of pome and stone fruits that causes severe economic losses each year. However, little is known about inoculum flow within or between orchards and pathogen establishment in an orchard, because few methods exist for detecting diversity or tracking isolates over time. SSR loci are an effective option, but may be confounded by a high degree of mutability and potential sensitivity to abiotic stress. Through transcriptome analysis, we identified novel markers mrr1, DHFR and MfCYP01 and validated stability of these markers under fungicide stress in natural infection sites. Nucleotide variation within mrr1, DHFR and MfCYP01 sequences differentiated isolates at all spatial scales: within the same infection site, between trees and between two farms. Sequenced regions were also effective for matching isolates collected from blossoms at the beginning of the season to progeny in cankers obtained at the end of the season. Collectively, results show that mrr1, DHFR and MfCYP01 are able to accurately differentiate M. fructicola isolates at the population level, can be used to track isolates over time, and are more stable than SSRs under external stresses. Either by themselves or combined with SSR markers, these gene-encoding regions are a much-needed tool for better understanding M. fructicola population dynamics. © 2017 Society of Chemical Industry.

Effects of Sublethal Fungicides on Mutation Rates and Genomic Variation in Fungal Plant Pathogen, Sclerotinia sclerotiorum.

Pathogen exposure to sublethal doses of fungicides may result in mutations that may represent an important and largely overlooked mechanism of introducing new genetic variation into strictly clonal populations, including acquisition of fungicide resistance. We tested this hypothesis using the clonal plant pathogen, Sclerotinia sclerotiorum. Nine susceptible isolates were exposed independently to five commercial fungicides with different modes of action: boscalid (respiration inhibitor), iprodione (unclear mode of action), thiophanate methyl (inhibition of microtubulin synthesis) and azoxystrobin and pyraclostrobin (quinone outside inhibitors). Mycelium of each isolate was inoculated onto a fungicide gradient and sub-cultured from the 50–100% inhibition zone for 12 generations and experiment repeated. Mutational changes were assessed for all isolates at six neutral microsatellite (SSR) loci and for a subset of isolates using amplified fragment length polymorphisms (AFLPs). SSR analysis showed 12 of 85 fungicide-exposed isolates had a total of 127 stepwise mutations with 42 insertions and 85 deletions. Most stepwise deletions were in iprodione- and azoxystrobin-exposed isolates (n = 40/85 each). Estimated mutation rates were 1.7 to 60-fold higher for mutated loci compared to that expected under neutral conditions. AFLP genotyping of 33 isolates (16 non-exposed control and 17 fungicide exposed) generated 602 polymorphic alleles. Cluster analysis with principal coordinate analysis (PCoA) and discriminant analysis of principal components (DAPC) identified fungicide-exposed isolates as a distinct group from non-exposed control isolates (PhiPT = 0.15, P = 0.001). Dendrograms based on neighbor-joining also supported allelic variation associated with fungicide-exposure. Fungicide sensitivity of isolates measured throughout both experiments did not show consistent trends. For example, eight isolates exposed to boscalid had higher EC50 values at the end of the experiment, and when repeated, only one isolate had higher EC50 while most isolates showed no difference. Results of this support the hypothesis that sublethal fungicide stress increases mutation rates in a largely clonal plant pathogen under in vitro conditions. Collectively, this work will aid our understanding how non-lethal fungicide exposure may affect genomic variation, which may be an important mechanism of novel trait emergence, adaptation, and evolution for clonal organisms.

Azoxystrobin and propiconazole offer significant potential for rice blast (Pyricularia oryzae) management in Australia

Fungicides are the preferred rice blast (Pyricularia oryzae) control option by farmers. However, no fungicides are yet registered for this purpose in Australia. Hence, it is important to test the baseline sensitivity of P. oryzae isolates collected from blast-affected regions across northern Australia, which have not yet been exposed to the fungicides, as part of a resistance management strategy. Further, it is also important to investigate and compare effect of application timing of fungicides on conidial development, including germination and germ tube growth, and penetration on susceptible rice. The EC50 of a collection of fungicide-sensitive blast isolates were within the range of 0.02–2.02 and 0.06–1.91 mg L−1 for azoxystrobin and propiconazole, respectively. Azoxystrobin was shown to have greater inhibitory effect on conidial germination than propiconazole. In addition, for pre-inoculation application, only germ tubes in the presence of external nutrients continued to grow from 24 to 48 hpi. On susceptible seedlings, both fungicides completely controlled blast disease when applied the same day as inoculation. However, for pre- or post-inoculation application of fungicide, the extent of disease control was reduced, with azoxystrobin more efficacious than propiconazole. A stimulatory effect of both fungicides at low dose was observed on certain P. oryzae isolates. This is the first study to assess the baseline sensitivity of the P. oryzae population in Australia and the first to report a stimulatory effect of low azoxystrobin concentration on growth of P. oryzae. The study highlights, for the first time, the critical role of external nutrients in promoting germ tube growth under fungicide stress conditions. Lastly, it demonstrates the high degree of efficacy of the fungicides and their potential for future rice blast management in Australia.

Effect of fungicides on plant growth promoting activities of phosphate solubilizing Pseudomonasputida isolated from mustard (Brassica compestris) rhizosphere

This study was navigated to examine the effects of fungicide-stress on the activities of plant-growth-promoting rhizobacterium Pseudomonasputida with inherent phosphate solubilizing activity. The fungicide-tolerant and phosphate solubilizing P.putida strain PS9 was isolated from the mustard rhizosphere and tentatively identified following standard morphological, physiological and biochemical tests. To further consolidate the identity of the strain PS9, the 16S rDNA sequence analysis was performed. Following the BLAST program, the strain PS9 was identified as P.putida. In the presence of the varying concentrations (0–3200μgmL−1; at a two fold dilution interval) of four fungicides of different chemical families (tebuconazole, hexaconazole, metalaxyl and kitazin) amended in minimal salt agar medium, the P.putida strain PS9 showed a variable tolerance levels (1400–3200μgmL−1) against the tested fungicides. The strain PS9 produced plant-growth-promoting (PGP) substances in significant amount in the absence of fungicides. In general, fungicides applied at the recommended, two and three times of the recommended rates, decreased the PGP attributes of P.putida the strain PS9 and affected the PGP activities in concentration-dependent manner. Fungicides at the recommended dose had minor reducing effect while the doses higher than the recommended dose significantly reduced the PGP activities (phosphate solubilization, salicylic acid, 2,3-dihydroxy benzoic acid, and indole-3-acetic acid production except exo-polysaccharides, hydrogen cyanate and ammonia production). Of the four fungicides, tebuconazole generally, showed maximum toxicity to the PGP activities of the strain PS9. This study inferred that fungicides must be examined in vitro for their possible adverse effects on soil micro flora before their application in agricultural fields. Moreover, the results also suggested the prerequisite of application of fungicide-tolerant PGPR strains as bioinoculants so that their PGP activities may not be suppressed under fungicide stress.

The adaptive potential of a plant pathogenic fungus, Rhizoctonia solani AG-3, under heat and fungicide stress

The ability to improve fitness via adaptive evolution may be affected by environmental change. We tested this hypothesis in an in vitro experiment with the plant pathogen Rhizoctonia solani Anastomosis Group 3 (AG-3), assessing genetic and environmental variances under two temperatures (optimal and higher than optimal) and three fungicide concentrations (no fungicide, low and high concentration of a copper-based fungicide). We measured the mean daily growth rate, the coefficient of variation for genotypic (I (G)) and environmental variance (I (E)) in growth, and broad-sense heritability in growth. Both higher temperature and increased fungicide concentration caused a decline in growth, confirming their potential as stressors for the pathogen. All types of standardized variances in growth-I (G), phenotypic variance, and I (E) as a trend-increased with elevated stress. However, heritability was not significantly higher under enhanced stress because the increase in I (G) was counterbalanced by somewhat increased I (E). The results illustrate that predictions for adaptation under environmental stress may depend on the type of short-term evolvability measure. Because mycelial growth is linked to fitness, I (G) reflects short-term evolvability better than heritability, and it indicates that the evolutionary potential of R. solani is positively affected by stress.

Development of a novel multiplex DNA microarray for Fusarium graminearum and analysis of azole fungicide responses

BackgroundThe toxigenic fungal plant pathogen Fusarium graminearum compromises wheat production worldwide. Azole fungicides play a prominent role in controlling this pathogen. Sequencing of its genome stimulated the development of high-throughput technologies to study mechanisms of coping with fungicide stress and adaptation to fungicides at a previously unprecedented precision. DNA-microarrays have been used to analyze genome-wide gene expression patterns and uncovered complex transcriptional responses. A recently developed one-color multiplex array format allowed flexible, effective, and parallel examinations of eight RNA samples.ResultsWe took advantage of the 8 × 15 k Agilent format to design, evaluate, and apply a novel microarray covering the whole F. graminearum genome to analyze transcriptional responses to azole fungicide treatment. Comparative statistical analysis of expression profiles uncovered 1058 genes that were significantly differentially expressed after azole-treatment. Quantitative RT-PCR analysis for 31 selected genes indicated high conformity to results from the microarray hybridization. Among the 596 genes with significantly increased transcript levels, analyses using GeneOntology and FunCat annotations detected the ergosterol-biosynthesis pathway genes as the category most significantly responding, confirming the mode-of-action of azole fungicides. Cyp51A, which is one of the three F. graminearum paralogs of Cyp51 encoding the target of azoles, was the most consistently differentially expressed gene of the entire study. A molecular phylogeny analyzing the relationships of the three CYP51 proteins in the context of 38 fungal genomes belonging to the Pezizomycotina indicated that CYP51C (FGSG_11024) groups with a new clade of CYP51 proteins. The transcriptional profiles for genes encoding ABC transporters and transcription factors suggested several involved in mechanisms alleviating the impact of the fungicide. Comparative analyses with published microarray experiments obtained from two different nutritional stress conditions identified subsets of genes responding to different types of stress. Some of the genes that responded only to tebuconazole treatment appeared to be unique to the F. graminearum genome.ConclusionsThe novel F. graminearum 8 × 15 k microarray is a reliable and efficient high-throughput tool for genome-wide expression profiling experiments in fungicide research, and beyond, as shown by our data obtained for azole responses. The array data contribute to understanding mechanisms of fungicide resistance and allow identifying fungicide targets.

Refining current knowledge on the yeast FLR1 regulatory network by combined experimental and computational approaches

Multidrug resistance is often the result of the activation of drug efflux pumps able to catalyze the extrusion of the toxic compound to the outer medium, this activation being frequently controlled at the transcriptional level. Transcriptional regulation in the model eukaryote S. cerevisiae is the result of the interaction and cross-talk between networks of transcription factors. This is the case of the transcriptional activation of the FLR1 gene occurring in response to stress induced by the agricultural fungicide mancozeb in yeast. FLR1 up-regulation depends on the integrated action of Yap1, a key regulator of oxidative stress response, Pdr3 and Yrr1, two of the transcription factors controlling multidrug resistance, and Rpn4, a regulator of proteasome gene expression, which interplay to produce the observed transcriptional up-shift. Based on the expression profiles of FLR1, YAP1, PDR3, YRR1 and RPN4 registered during yeast adaptation to stress induced by mancozeb and using a qualitative modeling approach, a model of the FLR1 regulatory network was built, and the response of S. cerevisiae to mancozeb stress was simulated. The use of a qualitative approach is especially useful to overcome the lack of enough quantitative data on kinetic parameters and molecular concentrations, permitting the immediate focus on the qualitative behavior of the system. This Systems Biology approach allowed the identification of essential features of the early yeast response to fungicide stress. The resulting model allowed the formulation of new hypotheses, in a quick and cost effective manner, on the qualitative behavior of the system following mancozeb challenge, some of which were validated experimentally. In particular, Pdr3 and Yrr1 were shown to directly control FLR1 up-regulation in mancozeb-challenged cells, based on the analysis of the effect of the inactivation of their putative binding sites in the FLR1 promoter. Furthermore, the inter-dependent role of Yap1 and Yrr1 in the regulation of PDR3 and RPN4 was brought to light, this joint activity possibly being extensible to eight other genes involved in multidrug resistance. The FLR1 network structure was revised, based on the comparison between simulated and experimental gene expression data in the double deletion mutant strains Δyrr1Δpdr3 and Δyrr1Δrpn4, and an additional, still unidentified, transcription factor was found to be required to fully explain the behavior of the network.

The Perspective Effects of Various Seed Coating Substances on Rice Seed Variety Khao Dawk Mali 105 Storability II: The Case Study of Chemical and Biochemical Properties

The aim of this study was to investigate the effects of seed coating substances; chemical fungicide (CA) and biological fungicide polymers [chitosan-lignosulphonate polymer (CL) and eugenol incorporated into chitosan-lignosulphonate polymer (E+CL)] on chemical and biochemical changes of rice seeds cv. KDML 105, which have been studied during storage for 12 months. CA significantly affected the rice seed chemical properties and the associated seed deterioration. After 12 months storage, protein content decreased accompanied by declined of lipid content, increased free fatty acids and activated lipoxygenase enzyme. In the case of biological fungicide coated seeds, the antioxidative scavenging enzymes were ascorbate peroxidase and superoxide dismutase and a high antioxidant activity protected them. Moreover, the sugar content was positive correlated with seed germination and vigor. The biological coated seeds were found to maintain high sugar contents inside the seeds, which resulted high seed storability significantly. In contrast, under fungicide stress (CA), those compounds were lost that directly affected seed vigor during storage.

Botryticides affect grapevine leaf photosynthesis without inducing defense mechanisms

The effects of the two botryticides, fludioxonil (fdx) and fenhexamid (fhd), were investigated on grapevine leaves (Vitis vinifera L. cv. Pinot noir) following photosynthesis and defense mechanisms. Treatments were carried out in vineyard at the end of flowering. Phytotoxicity of both fungicides was evaluated by measuring variations of leaf photosynthetic parameters and correlated expression of photosynthesis-related genes. Results demonstrated that similar decrease in photosynthesis was caused by fdx and fhd applications. Moreover, the mechanism leading to photosynthesis alteration seems to be the same for both fungicides. Stomatal limitation to photosynthetic gas exchange did not change following treatments indicating that inhibition of photosynthesis was mostly attributed to non-stomatal factors. Nevertheless, fungicides-induced depression of photosynthesis was related neither to a decrease in Rubisco carboxylation efficiency and in the capacity for regeneration of ribulose 1,5-bisphosphate nor to loss in PSII activity. However, fdx and fhd treatments generated repression of genes encoding proteins involved in the photosynthetic process. Indeed, decreased photosynthesis was coupled with repression of PsbP subunit of photosystem II (psbP1), chlorophyll a/b binding protein of photosystem I (cab) and Rubisco small subunit (rbcS) genes. A repression of these genes may participate in the photosynthesis alteration. To our knowledge, this is the first study of photosynthesis-related gene expression following fungicide stress. In the meantime, defense responses were followed by measuring chitinase activity and expression of varied defense-related genes encoding proteins involved in phenylpropanoid synthesis (PAL) or octadecanoid synthesis (LOX), as well as pathogenesis-related protein (Chi4C). No induction of defense was observed in botryticides-treated leaves. To conclude, the photosynthesis is affected without any triggering of plant defense responses.

Mechanisms and significance of fungicide resistance.

In this review article, we show that occurrence of fungicide resistance is one of the most important issues in modern agriculture. Fungicide resistance may be due to mutations of genes encoding fungicide targets (qualitative fungicide resistance) or to different mechanisms that are induced by sub-lethal fungicide stress. These mechanisms result in different and varying levels of resistance (quantitative fungicide resistance). We discuss whether or not extensive use of fungicides in agricultural environments is related to the occurrence of fungicide resistance in clinical environments. Furthermore, we provide recommendations of how development of fungicide resistant pathogen populations may be prevented or delayed.

Plant and soil response to single and mixed species of arbuscular mycorrhizal fungi under fungicide stress

Defining sustainable agricultural practices requires an understanding of both above- and below-ground consequences to management strategies. While alternatives to biocides are sought for the control of weeds, insects and pathogens, biocide use will continue with the goal of reducing quantities used in an integrated approach. The impact of three fungicides on plant growth, seed yield, seed nutrient composition, and on soil aggregation as mediated by arbuscular mycorrhizal (AM) fungi was studied in a silty-clay loam soil with a high extractable P concentration. Shoot dry mass, seed yield and seed nutrient (N, P, K) contents of pea ( Pisum sativum L.) plants were enhanced by three AM fungi and a mixture of three fungi compared with nonAM plants. Each AM fungus produced a distinct pattern of shoot responses of plants, but the mixed inoculum treatment was as good or better than each single species for all of the above-ground measures of plant performance. Soil aggregation was improved by two of the three AM fungi as individual inocula, and was further increased in the mixed inoculum treatment. Two of the three fungicides reduced shoot dry mass and seed yield, but none of the fungicides affected soil aggregation. Fungicides inhibited mycorrhiza formation least in the mixed inoculum treatment which gave the best overall plant and soil responses. Since the three fungi together were more tolerant of fungicides than each fungus alone, it appeared that as a community, AM fungi modified and alleviated fungicide stress, resulting in high levels of plant performance and soil aggregation.

DMI-Resistance in Ustilago maydis: 1. Characterization and Genetic Analysis of triadimefon-resistant laboratory mutants

Triadimefon-resistant Ustilago maydis mutants were obtained by UV-irradiation and by selection of spontaneous mutants during successive adaptation. Resistance factors varied considerably among the isolates ranging from 2 to 42. The UV-induced mutants only exhibited low resistance levels, but resistance could be raised by repeated UV-irradiation. Resistance in most of the mutants decreased after repeated subculturing in fungicide-free liquid medium. The spontaneous mutants were cross-resistant, whereas some UV-induced mutants were not cross-resistant, to fenarimol, nuarimol, and imazalil. The fitness of the resistant strains was not different from that of the parental strains. No differences in virulence and sporidial morphology were detected between resistant and sensitive strains. However, growth rates were slightly reduced in liquid culture. The low resistance levels of the UV-induced mutants turned out not to be of advantage under fungicide stress. Genetic analysis of crosses between resistant and sensitive strains showed that triadimefon-resistance is chromosomally fixed and probably under polygenic control. The spontaneous mutants are suggested to be suitable model systems for further studies concerning the resistance mechanism of sterol demethylation inhibitors.