Pathogen DNA Content Research Articles

Identification of a bio-signature for barley resistance against Pyrenophora teres infection based on physiological, molecular and sensor-based phenotyping

Necrotic and chlorotic symptoms induced during Pyrenophora teres infection in barley leaves indicate a compatible interaction that allows the hemi-biotrophic fungus Pyrenophora teres to colonise the host. However, it is unexplored how this fungus affects the physiological responses of resistant and susceptible cultivars during infection. To assess the degree of resistance in four different cultivars, we quantified visible symptoms and fungal DNA and performed expression analyses of genes involved in plant defence and ROS scavenging. To obtain insight into the interaction between fungus and host, we determined the activity of 19 key enzymes of carbohydrate and antioxidant metabolism. The pathogen impact was also phenotyped non-invasively by sensor-based multireflectance and –fluorescence imaging.Symptoms, regulation of stress-related genes and pathogen DNA content distinguished the cultivar Guld as being resistant. Severity of net blotch symptoms was also strongly correlated with the dynamics of enzyme activities already within the first day of infection. In contrast to the resistant cultivar, the three susceptible cultivars showed a higher reflectance over seven spectral bands and higher fluorescence intensities at specific excitation wavelengths. The combination of semi high-throughput physiological and molecular analyses with non-invasive phenotyping enabled the identification of bio-signatures that discriminates the resistant from susceptible cultivars.

Tissue specific colonization of Phytophthora capsici in Capsicum spp.: molecular insights over plant-pathogen interaction

Root and crown rot (RCR) caused by Phytophthora capsici is present in all crop production areas of pepper and chili worldwide. This pathogen was recently reported at the Pacific coast of Ecuador (province of Manabi), as an etiological agent of wilt and root and crown rot in commercial fields of bell pepper (Capsicum annuum). This study aimed to evaluate the resistance of ten Capsicum spp. genotypes artificially infected with P. capsici and to compare the tissue-specific colonization of P. capsici under greenhouse conditions. Disease intensity, mycelial growth incidence and pathogen DNA content in root and hypocotyl tissues of P. capsici infected Capsicum germplasm was evaluated at 17 days after infection (dai). Results indicated that genotypes Nathalie, ECU-12831, ECU-9129, Codigo 5, and ECU-1296 were found to be resistant, and ECU-11995, ECU-2254 B, California Wonder, Quetzal and Marcato showed susceptibility to the disease. The amount of pathogen DNA in roots was the only variable that did not correlate with the resistance or susceptibility of the genotypes. In fact, in resistant genotypes the pattern of pathogen DNA content was higher in roots than hypocotyls (but also in the susceptible ECU-11995), while in the susceptible genotypes (e.g. ECU-2254 B, C. Wonder, Quetzal and Marcato) showed the opposite pattern. The present work provides new insights of host-pathogen interaction, which might be used in breeding programs aiming to develop Capsicum cultivars with resistance to RCR.

Reaction of winter wheat cultivars to eyespot assessed visually and by real-time PCR

The reaction of twelve winter wheat cultivars frequently grown in the Czech Republic and twenty-five new breeding lines to inoculation with Oculimacula yallundae and Oculimacula acuformis was evaluated in small plot trials from 2017–2018. The assessment was carried out visually by symptoms and by a quantitative real-time polymerase chain reaction (qPCR). The aims of the study were to compare the results of both methods, to evaluate the effect of the resistance gene Pch1 to eyespot, and to select new breeding lines resistant to eyespot. The relationship between the eyespot symptoms and the pathogen DNA content in plant tissues followed a moderate linear regression. Low levels of eyespot were observed in the cultivars/lines possessing the resistance gene Pch1 (Annie, Hermann, Rebell, SG-S1215-14, SG-S1825-14, SG-S791-13) and also in the line SG-SU630-15. The qPCR method was able to detect low levels of the pathogens in the plant tissue and to distinguish two eyespot pathogens. O. acuformis was detected in very low concentrations in the inoculated plants compared with O. yallundae. The eyespot infection rate was significantly higher in 2017 than in the next agricultural season due to extremely dry and warm spring weather in 2018.

Genome-wide identification and expression analysis of chitinase gene family in Brassica rapa reveals its role in clubroot resistance

Chitinases, a category of pathogenesis-related proteins, are responsible for catalyzing the hydrolysis of chitin into the N-acetyl-d-glucosamine. Therefore, chitinases are believed to function as a guardian against chitin-containing pathogens. Here, we examined the role of the Brassica rapa chitinase family genes in clubroot disease. A total of 33 chitinase genes were identified and grouped into five classes based on their conserved domain. They were distributed unevenly across eight chromosomes in B. rapa, and 31 of them contained few introns (≤2). In addition, the expression of these genes was organ-specific, and 14 genes were expressed differentially in response to Plasmodiophora brassicae challenge of clubroot-susceptible (CS NIL) and resistant (CR NIL) lines. Furthermore, reduced pathogen DNA content and clubroot symptoms were observed in the CS NILs after their treatment with chitin oligosaccharides 24 h prior to inoculation with P. brassicae. The findings indicate that chitinases play a crucial role in pathogen resistance of the host plants. The results offer an insight into the role of chitinase in B. rapa–P. brassicae interaction.

Starch Hydrolysis and Vessel Occlusion Related to Wilt Symptoms in Olive Stems of Susceptible Cultivars Infected by Verticillium dahliae.

This study investigated starch content, amount of pathogen DNA and density of occluded vessels in healthy and Verticillium dahliae infected olive shoots and stems. Starch hydrolysis is considered a mechanism to refill xylem vessels that suffered cavitation by either, drought conditions or pathogen infections. The main objective of this work was to evaluate this mechanism in olive plants subjected to V. dahliae infection or to drought conditions, in order to know the importance of cavitation in the development of wilting symptoms. In initial experiments starch content in the shoots was studied in trees of cultivars differing in the level of resistance growing in fields naturally infested with V. dahliae. The starch content, esteemed by microscopic observation of stem transversal sections stained with lugol, decreased with the level of symptom severity. Results were confirmed in a new experiment developed with young plants of cultivars ‘Picual’ (highly susceptible), ‘Arbequina’ (moderately susceptible) and ‘Frantoio’ (resistant), growing in pots under greenhouse conditions, either inoculated or not with V. dahliae. In this experiment, the pathogen DNA content, quantified by real-time PCR, and the density of occluded vessels, recorded by microscopic observations of transversal sections stained with toluidine blue, were related to the symptoms severity caused by the pathogen. Finally, a drought experiment was established with young plants of the cultivar ‘Picual’ grown in pots under greenhouse conditions in order to compare the effects caused by water deficit with those caused by the pathogen infection. In both cases, results show that starch hydrolysis occurred, what indirectly evidence the importance of xylem cavitation in the development of the symptoms caused by V. dahliae but in the water stressed plants no vessel occlusion was detected.

Modeling Pathogen DNA Content and Visual Disease Assessment in Seed Tubers to Inform Disease in Potato Progeny Root, Stolon, and Tubers.

Measurement of pathogens on seed tubers is essential for informing likelihood of subsequent potato disease. Here we utilized quantitative PCR assessment of pathogen DNA and visual assessment of disease to measure seed tuber inoculum and used this to model development of disease in potato grown in pathogen-free soil. Analysis by recursive partitioning and modeling using receiver operating curves indicated both abundance of Rhizoctonia solani AG3 and Streptomyces scabies DNA, and disease symptoms associated with these pathogens on seed tubers could predict subsequent disease in progeny tubers and for R. solani, stolons. In contrast, abundance of Spongospora subterranea DNA and disease symptoms on seed tubers were not consistently associated with powdery scab in progeny tubers. The relationship between S. subterranea DNA and seed tuber symptoms on root galling was stronger. Symptomless seed tubers that carried high levels of S. subterranea DNA were also associated with greater root galling than those with low pathogen DNA levels. There was a modest association between root galling and powdery scab in progeny tubers. These results highlight the importance of using certified seed tubers, and demonstrate a statistical tool for measuring the impact of seed tuber-borne inoculum.

A comparison of potato seed-tuber sampling strategies using visual and DNA analyses to estimate incidence of major seed tuber-borne pathogens

Potato seed certification is a disease management tool that minimises the risk of spreading seed tuber-borne inoculum of infectious diseases. Traditionally, certification sampling strategies have relied upon visual assessment of a seedlot from samples taken at one or two points within the load of seed tubers. However methodologies in selection of tuber samples have not been critically assessed for their precision in estimating disease load. This study presents an analysis of 37 potato seedlots over a 3 year period. Analysis of sample data using receiver operating curves (ROCs) indicates that point sampling taking two samples of 100 tubers at the beginning and end of a seedlot gives equivalent disease estimation as a continuous sampling strategy taking ten samples of 20 tubers randomly throughout the seedlot, although at lower statistical precision. This was confirmed both by visual assessment of tuber-borne disease and by analysis of pathogen DNA content from tuber peel. Across the 3 years of study, powdery scab and black scurf were the major seed tuber-borne diseases recognised and this corresponded with high levels of pathogen DNA from peel analysis for both Spongospora subterranea and Rhizoctonia solani AG3 respectively.

A Real-Time PCR Assay for Detection and Quantification of Verticillium dahliae in Spinach Seed

Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.

Development of a real-time PCR method for the identification of wheat genotypes carrying different eyespot resistance genes

With 2 figures and 7 tables Abstract Eyespot, caused by Oculimacula acuformis and O. yallundae, is an important disease of wheat (Triticum aestivum). The two pathogens generate similar symptoms and cannot be distinguished visually. In order to distinguish O. acuformis and O. yallundae infections and to identify reliable quantitative differences in the level of resistance of wheat genotypes, two SYBR Green based real-time PCR assays were developed. The influence of time after inoculation until sample collection, the effect of species (O. acuformis, O. yallundae), and the relevance of wheat cultivars carrying Pch1, Pch2, or none of the resistance genes on the pathogen-DNA content were analysed. The most clear-cut difference of pathogen content between resistant and susceptible wheat genotypes was obtained 12 weeks after inoculation with O. yallundae. Growth chamber tests using artificial inoculation with O. yallundae and sampling 12 weeks after inoculation for real-time PCR is shown to be a reliable procedure to determine the resistance level of wheat against eyespot. Likewise, this is the basis for the development of efficient molecular markers.

Effects of genotype, environment and fungicide treatment on development of Fusarium head blight and accumulation of DON in winter wheat grain

Reactions to artificial infection with <i>Fusarium culmorum</i> and (metconazole- or tebuconazole-based) fungicides were studied in nine winter wheat cultivars that were evaluated in field experiments at the location Prague-Ruzyne for four years (2001−2004) for deoxynivalenol (DON) content in grain, pathogen DNA content (Ct) by real-time quantitative PCR, percentage of Fusarium damaged grains (FDG), symptom scores and reductions in grain yield components. All examined traits were highly affected by conditions of experimental years and interactions with cultivars and treatments. Moderately resistant cultivars Arina and Petrus were included in the first homogeneous group in all traits, including the pathogen DNA content. To predict cultivar resistance to Fusarium head blight and accumulation of DON, the examination of the percentage of FDG in different environments appeared to be useful from practical aspects. The pathogen DNA content was significantly related to the content of DON under different conditions, however, the correlation coefficients ranged between 0.42 and 0.92. Different levels of DON could be detected at similar pathogen contents. The higher colonization of grain by the fungus was mostly connected with a strongly reduced amount of DON per pathogen unit (DON/Ct ratio). The fungicide treatment had a significant effect on a reduction in all traits except DON/Ct, but the effects on different traits were not often proportional and they were highly variable in the particular years (range 10−69%) and cultivars (range < 0−60%). While the application of fungicide caused a reduction in DON content in all cultivars, an increase in pathogen content after the application of fungicides was not exceptional. The low fungicide effect on a reduction in pathogen content was connected with higher temperatures (temperature extremes) in a 30-day period of disease development. The efficacy of fungicide treatment for DON was low at high pathogen content and late heading. The use of the collected data to improve control measures is discussed.

Use of real-time PCR to examine the relationship between disease severity in pea and Aphanomyces euteiches DNA content in roots

Aphanomyces euteiches causes severe root rot of peas. Resistance is limited in commercial pea cultivars. Real-time fluorescent PCR assay specific for A. euteiches was used to study the relationship between disease severity and pathogen DNA content in infected peas. Five pea genotypes ranging in levels of resistance were inoculated with five isolates of A. euteiches. Plants were visually rated for disease development and the amount of pathogen DNA in roots was determined using the PCR assay. The susceptible genotypes Genie, DSP and Bolero tended to have significantly more disease and more pathogen DNA than the resistant genotypes 90-2079 and PI 180693. PI 180693 consistently had less disease, while 90-2079 had the lowest amount of pathogen DNA. The Spearman correlation between pathogen DNA quantity and disease development was positive and significant (P < 0.05) for three isolates, but was not significant for two other isolates. This suggests that the real-time PCR assay may have limited application as a selection tool for resistance in pea to A. euteiches. Its utility as a selection tool would be dependent on the correlation between disease development and pathogen DNA content for a given pathogen isolate. The accuracy and specificity of the real-time PCR assay suggests considerable application for the assay in the study of mechanisms of disease resistance and the study of microbial population dynamics in plants.

Quantifying Phytophthora medicaginis in Susceptible and Resistant Alfalfa with a Real‐Time Fluorescent PCR Assay

AbstractA real‐time fluorescent PCR assay using a set of specific primers and a fluorochrome‐labelled probe (TaqMan) was developed to quantify the amount of Phytophthora medicaginis DNA in alfalfa plants that were classified as either resistant or susceptible to the pathogen based on visual assessment of disease response. The assay clearly discriminated among three standard check alfalfa populations with different levels of resistance based on the analysis of DNA extracted from the roots of bulked plant samples. In two independent experiments, the Spearman rank correlation between pathogen DNA content and the number of susceptible plants in a bulked sample was greater than 0.89 and highly significant (P&lt;0.0001). Significantly less pathogen DNA was detected in bulked samples of a highly resistant check population than in bulked samples from more susceptible check populations. Analysis of individual plants indicated that significantly less pathogen DNA was detected in resistant plants than in susceptible plants. Applications of the assay are considered for breeding programs and the study of microbial population dynamics in plants simultaneously infected with different pathogens.

Quantitative monitoring of colonization of olive genotypes by Verticillium dahliae pathotypes with real-time polymerase chain reaction

For Verticillium wilt diseases, quantitative measurement of the pathogen in infected tissues is necessary for characterizing the disease reaction of host genotypes to pathogen strains. The amount of pathogen DNA in the plant should be representative of the number of potential propagules. In this work, we quantified the DNA of highly virulent, defoliating (D) and mildly virulent, nondefoliating (ND) Verticillium dahliae in Verticillium wilt resistant (Acebuche-L), and -susceptible (Arbequina and Picual) olive genotypes by real-time polymerase chain reaction (PCR) assays. We show that the amount of pathogen DNA quantified in each olive genotype correlated with susceptibility to Verticillium wilt, but was influenced less by virulence of the infecting V. dahliae pathotype, suggesting that the extent of pathogen colonization does not clearly determine the virulence phenotype. Maximal pathogen DNA occurred in root and stem tissues before disease had fully developed in the susceptible olive genotypes, with pathogen DNA content in stem tissue being lower than that in root tissues. A boost in V. dahliae propagules in root tissues, as indicated by the amount of D and ND DNA, took place 1 week after inoculation, followed by a decrease over time. That decrease was sharp in Arbequina and Acebuche-L plants compared with a progressive reduction in Picual. The amount of V. dahliae DNA in roots of Picual plants remained at a high level for a long period of time following inoculation, but it eventually decreased too. Similar changes in pathogen DNA amounts over time occurred also in stem. Verticillium wilt hardly developed in Acebuche-L plants, but the amount of D and ND V. dahliae DNA in roots was comparable to that found in Arbequina plants. However, pathogen DNA could not be quantified in stems of Acebuche-L plants though it was detectable in them by nested-PCR. The results in our study show that real-time PCR is an excellent tool for quantitative diagnosis of olive infection by D and ND V. dahliae; as well as for monitoring pathogen colonization and assessing resistance and tolerance to Verticillium wilt in olive genotypes. This would help in olive breeding programs for resistance to the disease.

Quantifying the relationship between disease severity and the amount of Aphanomyces euteiches detected in roots of alfalfa and pea with a real-time PCR assay

A real-time PCR assay was used to quantify the relationship in alfalfa and pea between disease severity and the amount of Aphanomyces euteiches detected in roots. The study included isolates of race 1 and race 2 of the alfalfa pathovar of A. euteiches and an isolate obtained from diseased pea. Spearman rank correlations between pathogen DNA content and disease severity index (DSI) ratings were positive ( ⩾̸ 0.57) and significant (P ⩽ 0.0007) for individual alfalfa plants, bulked alfalfa plant samples, and individual pea plants. In all experiments, significantly more pathogen was detected in susceptible populations than in resistant populations. The results clearly demonstrate that resistance to A. euteiches in both alfalfa and pea is characterized by a reduction in pathogen colonization relative to levels observed for susceptible reactions. The assay was very specific for A. euteiches, producing very linear assays with DNA extracted from pathogen isolates obtained from alfalfa, pea, and bean. Possible applications of the assay in conjunction with other real-time PCR assays specific to other legume pathogens are discussed in relation to simultaneous disease screening for multiple plant pathogens and the study of microbial population dynamics in mixed plant infections.

QuantifyingAphanomyces euteichesin Alfalfa with a Fluorescent Polymerase Chain Reaction Assay

ABSTRACT A polymerase chain reaction (PCR) assay using a set of specific primers and a dual-labeled probe (TaqMan) was developed to quantify the amount of Aphanomyces euteiches DNA in alfalfa plants exhibiting varying levels of disease severity. The study included isolates of race 1 and race 2 of A. euteiches. The assay also discriminated between alfalfa populations for resistance based on analysis of DNA extracted from bulked plant samples. Analysis of individual plants and bulked plant samples of standard check populations with both pathogen isolates resulted in Spearman rank correlations between pathogen DNA content and disease severity index ratings that were greater than 0.75 and highly significant (P < 0.0005). In experiments with a race 1 isolate, the amount of pathogen DNA present in the resistant check WAPH-1 was significantly less than in the susceptible check Saranac. In experiments with a race 2 isolate, the amount of pathogen DNA in the resistant check WAPH-5 was significantly less than in either of the susceptible checks, Saranac and WAPH-1. Discrimination between commercial cultivars based on quantitative PCR analysis of bulked plant samples was similar to classification based on visual assessment of disease severity.