Polymerase Chain Reaction Detection of Ustilago hordei in Leaves of Susceptible and Resistant Barley Varieties

ABSTRACT Although Ustilago hordei infects barley seedlings, symptoms of the disease covered smut are not visible until heading. Natural or artificial inoculation usually results in inconsistent infection, even in highly susceptible lines. Thus, breeding for resistance to covered smut is time consuming and difficult. The ribosomal DNA internal transcribed spacer (ITS) regions of U. hordei were sequenced and a primer pair was developed for polymerase chain reaction (PCR). These primers amplified a 574-bp fragment from DNA of Ustilago spp., but did not amplify DNA from barley or other common barley pathogens. DNA extracted from as few as four U. hordei sporidia was detected by this method. U. hordei DNA was amplified from leaf tissue of inoculated susceptible and resistant plants at different stages of plant development in differential varieties. Growth of the fungus in different leaves of an individual plant was variable. Several highly resistant varieties were shown to contain U. hordei DNA in the first three to four leaves, but not in later leaves. Thus, although the fungus can infect many resistant plants, the plants remain symptomless. Detection of U. hordei prior to heading should assist efforts for breeding for resistance and provide clues concerning the mechanisms of resistance employed by different resistance genes.     

Development of a Real-Time Polymerase Chain Reaction Assay for Quantifying Verticillium albo-atrum DNA in Resistant and Susceptible Alfalfa

ABSTRACT A precise real-time polymerase chain reaction (PCR) assay was developed for quantifying Verticillium albo-atrum DNA. The assay was used in a repeated experiment to examine the relationship between the quantity of pathogen DNA detected in infected leaves and shoots and the severity of Verticillium wilt symptoms in several alfalfa cultivars expressing a range of disease symptoms. Plants were visually inspected for symptoms and rated using a disease severity index ranging from 1 to 5, and the quantity of pathogen DNA present in leaves and stems was determined with real-time PCR. No significant differences in pathogen DNA quantity or disease severity index were observed for experiments or for cultivar-experiment interactions. Significant differences were observed between cultivars for the quantity of pathogen DNA detected with real-time PCR and also for disease severity index ratings. In both experiments, the highly resistant check cultivar Oneida VR had significantly less pathogen DNA, and significantly lower disease severity index ratings than the resistant cultivar Samauri, the moderately resistant cultivar Vernema, and the susceptible check cultivar Saranac. In both experiments, the Spearman rank correlation between the amount of V. albo-atrum DNA detected in leaves and stems with real-time PCR and disease severity index ratings based on visual examination of symptoms was positive (>0.52) and significant (P < 0.0001). These results suggest that resistance to Verticillium wilt in alfalfa is characterized by a reduced colonization of resistant genotypes by the fungus.     

Quantification of Xylella fastidiosa from Citrus Trees by Real-Time Polymerase Chain Reaction Assay

ABSTRACT Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease of sweet orange cultivars in Brazil. Polymerase chain reaction (PCR)-based assays constitute the principal diagnostic method for detection of these bacteria. In this work, we established a real-time quantitative PCR (QPCR) assay to quantify X. fastidiosa in naturally and artificially infected citrus. The X. fastidiosa cell number detected in the leaves increased according to the age of the leaf, and bacteria were not detected in the upper midrib section in young leaves, indicating temporal and spatial distribution patterns of bacteria, respectively. In addition, the X. fastidiosa cell number quantified in leaves of 'Pera' orange and 'Murcott' tangor reflected the susceptible and resistant status of these citrus cultivars. None of the 12 endophytic citrus bacteria or the four strains of X. fastidiosa nonpathogenic to citrus that were tested showed an increase in the fluorescence signal during QPCR. In contrast, all 10 CVC-causing strains exhibited an increase in fluorescence signal, thus indicating the specificity of this QPCR assay. Our QPCR provides a powerful tool for studies of different aspects of the Xylella-citrus interactions, and can be incorporated into breeding programs in order to select CVC-resistant plants more quickly.     

Real-Time Polymerase Chain Reaction Quantification of Phytophthora capsici in Different Pepper Genotypes

ABSTRACT Reliable and sensitive quantification of Phytophthora capsici in pepper plants is of crucial importance in managing the multiple syndromes caused by this pathogen. A real-time polymerase chain reaction (PCR) assay was developed for the determination of P. capsici in pepper tissues. DNA levels of a highly virulent and a less virulent isolate were measured in different pepper genotypes with varying degrees of resistance. Using SYBR Green and specific primers for P. capsici, the minimal amount of pathogen DNA quantified was 10 pg. Pathogen DNA was recorded as early as 8 h postinoculation. Thereafter, the increase was rapid in susceptible cultivars and slower in resistant ones. The amount of pathogen DNA quantified in each pepper genotype correlated with susceptibility to Phytophthora root rot. Likewise, there was a relationship between the virulence of the pathogen and the degree of colonization. Differences also were found in oomycete amount among pepper tissues, with maximal pathogen biomass occurring in stems. The real-time PCR technique developed in this study was sensitive and robust enough to assess both pathogen development and resistance to Phytophthora root rot in different pepper genotypes.     

Barley yellow dwarf virus in ryegrass and its detection by ELISA

The enzyme-linked immunosorbent assay (ELISA) effectively detected PAV- and MAV-like strains of barley yellow dwarf virus (BYDV) in ryegrass. MAV-like BYDV was found in a large proportion of ryegrass plants with foliar symptoms. There was a poor association between foliar symptoms and PAV-like virus, which occurred with similar frequency in plants with and without symptoms. By August 1982, plots of perennial, Italian and hybrid ryegrass sown at Auchincruive in 1980 were extensively infected with PAV- and MAV-like strains of BYDV. Tests on samples from 1981- and 1982-sown plots in August 1983 also indicated early invasion by BYDV. Infection levels of 7–80% were found in 13 commercial crops of perennial ryegrass surveyed near Auchincruive in May 1983. PAV-like BYDV occurred with greater frequency than did MAV-like strains of the virus.     

Quantification of Magnaporthe grisea During Infection of Rice Plants Using Real-Time Polymerase Chain Reaction and Northern Blot/Phosphoimaging Analyses

ABSTRACT Rice blast, caused by Magnaporthe grisea, is a serious fungal disease of rice worldwide. Currently, evaluation of the fungal pathogenicity and host resistance is mainly based on a disease rating or measurement of blast lesion number and size. However, these methods only provide visual estimation rather than accurate measurement of fungal growth in rice plants. In this study, DNA-based real-time polymerase chain reaction (PCR) and RNA-based northern blot/phosphoimaging analyses were evaluated to quantify M. grisea. Both methods were sensitive, specific, and reproducible and could accurately measure the relative growth and absolute biomass of M. grisea. The real-time PCR analysis showed that the growth of M. grisea in seedling leaves of susceptible cultivars (M201 and Wells) was approximately 46 to 80 times higher than that of a resistant cultivar (Drew) at 4 and 6 days after inoculation. The data obtained from the real-time PCR assays also were consistent with that from northern blot/ phosphoimaging analysis. However, the real-time PCR approach was much faster and more convenient in most cases. Therefore, it is an excellent tool for in planta quantification of M. grisea and can be used for reliable assessment of fungal pathogenicity and host resistance.     

The Basis for Thinopyrum-Derived Resistance to Cereal yellow dwarf virus

ABSTRACT Incorporation of Thinopyrum intermedium-derived resistance genes into improved wheat germ plasm generated a wheat substitution line (P29) which is completely resistant to Cereal yellow dwarf virus (CYDV). The undetectable CYDV titer in P29 led many to conclude that resistance prevented viral replication. To determine whether CYDV replication or movement is inhibited, we examined inoculated leaves for replication and uninoculated leaves for systemic spread. CYDV subgenomic RNA, produced only during replication, was found within the inoculated area of P29 and T. intermedium leaves, demonstrating that viral replication occurred. Absence of CYDV from uninoculated, newly emerging leaves of inoculated P29 and T. intermedium plants indicated resistance via inhibition of viral systemic infection. Resistance was not effective if P29 was inoculated with 50 to 100 viruliferous aphids per plant at the first-leaf stage or younger, resulting in a systemic spread of CYDV. As these infected P29 seedlings continued to grow, the resistance phenotype was recovered. Our data suggested that T. intermedium-derived resistance to CYDV was primarily dosage dependent and could be developmentally regulated if the amount of inoculum was large enough.     

Detection and Quantification of Phytophthora ramorum from California Forests Using a Real-Time Polymerase Chain Reaction Assay

ABSTRACT The timely and accurate detection of pathogens is a critical aid in the study of the epidemiology and biology of plant diseases. In the case of regulated organisms, the availability of a sensitive and reliable assay is essential when trying to achieve early detection of the pathogen. We developed and tested a real-time, nested polymerase chain reaction (PCR) assay for the detection of Phytophthora ramorum, causal agent of sudden oak death. This technique then was implemented as part of a widespread environmental screen throughout California. The method here described is sensitive, detecting less than 12 fg of pathogen DNA, and is specific for P. ramorum when tested across 21 Phytophthora spp. Hundreds of symptomatic samples from 33 sites in 14 California counties were assayed, resulting in the discovery of 10 new host species and 23 infested areas, including 4 new counties. With the exception of a single host, PCR-based discovery of new hosts and infested areas always was confirmed by traditional pathogen isolations and inoculation studies. Nonetheless, molecular diagnostics were key in early pathogen detection, and steered the direction of further research on this newly discovered and generalist Phytophthora species.     

Identification and Quantification of Pathogenic Pythium spp. from Soils in Eastern Washington Using Real-Time Polymerase Chain Reaction

ABSTRACT Traditional methods of quantifying Pythium spp. rely on the use of selective media and dilution plating. However, high variability is inherent in this type of enumeration and counts may not be representative of the pathogenic population of Pythium spp. Variable regions of the internal transcribed spacer of the rDNA were used to design species-specific primers for detection and quantification of nine Pythium spp. from soils in eastern Washington. Primer pairs were designed for Pythium abappressorium, P. attrantheridium, P. heterothallicum, P. irregulare group I, P. irregulare group IV, P. paroecandrum, P. rostratifingens, P. sylvaticum, and P. ultimum and used with real-time polymerase chain reaction. Standard curves were generated for each of the species using SYBR Green I fluorescent dye for detection of amplification. Seventy-seven isolates of Pythium were screened to confirm specificity of each primer set. DNA was extracted from soil and standard curves were generated for P. irregulare group I, P. irregulare group IV, and P. ultimum to correlate populations of each species in the soil with quantities of DNA amplified from the same soil. Examination of raw field soils revealed results similar to those observed in previous studies. This new technique for the quantification of Pythium spp. is rapid and accurate, and will be a useful tool in the future study of these pathogenic Pythium spp.     

Analyzing and Modeling Temporal Disease Progress of Barley yellow dwarf virus Serotypes in Barley Fields

ABSTRACT Population dynamics of Padi avenae (PAV), Macrosiphum avenae (MAV), and Rhopalosiphum padi (RPV) virus serotypes of Barley yellow dwarf virus (BYDV) and of their main aphid vectors were studied in winter barley (Hordeum vulgare) fields for three successive years in western France. An epidemiological model of the spread of viruses in the field was developed based on vector populations as forcing variables and the population dynamics of each virus serotype. This model accurately simulated the kinetics of the epidemic for PAV serotypes, which are the most common ones. For RPV and to some extent for MAV, the results were less satisfactory. The occurrence and spread of PAV and MAV serotypes in the field was clearly and easily related to that of their main vector species. Conversely, the spread of RPV serotypes showed no consistent relationships with the dynamics of their vectors. Incidence of PAV in 1989 to 1990 and 1990 to 1991 showed a bimodal distribution, with maximums in fall (December) and spring (May) that were linked to fall infestations by R. padi and spring infestations by three (R. padi, Sitobion avenae, and Metopolophium dirhodum) or two (S. avenae and M. dirhodum) aphid species. In 1991 to 1992, the PAV infection curve was monomodal and mainly due to a primary spread of the virus by very large populations of alate R. padi. MAV incidence was low in fall and winter and reached a maximum in spring 1990 and 1991 related to the occurrence of S. avenae and M. dirhodum. RPV incidence was low every year, despite the abundance of its vector, R. padi. Mixed infections were more frequent than expected by chance and were assumed to be partly related to heterologous encapsidation. The occurrence of each serotype is discussed in relation to the time of crop infection and possible damage.     

Yield Effects of Barley yellow dwarf virus in Soft Red Winter Wheat

ABSTRACT Three cultivars of soft red winter wheat were evaluated to determine the relationship between the incidence and time of infection by Barley yellow dwarf virus (BYDV) and yield. Wheat was planted in 1995, 1996, and 1997 in a split-plot design with six replicates at sites in Indiana and Illinois. Yield plots were infested with different amounts of viruliferous aphids, and the incidence of BYDV in each plot was measured. In a 2-year study in Illinois with cv. Clark and the PAV-IL isolate of BYDV, yields were assessed following aphid infestation in fall, early spring, and late spring. Early spring infections resulted in larger yield reductions than late spring infections in both years and larger than fall infections in one year. Regression analyses to relate incidence of infection and yield with data from fall and early spring infections provided R(2) values of 0.89 and 0.51 for the 1996 to 1997 and 1997 to 1998 seasons, respectively. An additional study at the same site in the 1996 to 1997 season compared the yield responses of cvs. Clark, Y88-3e, and PT8935b. Increases in the incidence of BYDV correlated with decreases in yield, with R(2) values of 0.80, 0.78, and 0.90 for the three cultivars, respectively. Estimated yield losses in both studies and all cultivars ranged from 27 to 45 kg/ha or 0.34 to 0.55% for each percent increase in virus infection. In a third study over a 2-year period in Indiana with the same three wheat genot ypes and a second BYDV isolate (PAV-P), BYDV treatments resulted in significant reductions in yield, but yield loss and the incidence of BYDV were not linearly correlated. Given the differences in yield reductions caused by the two BYDV isolates, PAV-P may be an attenuated strain of BYDV and may cross-protect plants from naturally occurring strains of the virus.     

感染大麦黄矮病毒的小麦中vsiRNA特征分析

 RNA沉默是植物中一种保守的抗病毒机制,其以大量病毒来源的小干扰RNA(virus-derived small interfering RNAs,vsiRNAs)的产生为标志,病毒在侵染寄主过程中可通过vsiRNAs靶向寄主转录本以对抗这种防御机制。BYDV-GAV引起的小麦黄矮病导致小麦黄化和矮化症状,对小麦生产构成严重威胁。通过深度测序技术,分析了感染BYDV-GAV的感病小麦品种‘小偃6号’中vsiRNAs的特征,共得到11 384个vsiRNAs,并预测到37 784个寄主靶基因。发现来源于BYDV-GAV基因组的正义和反义链的vsiRNAs的数量分布大致相等,在5’末端具有A和C偏好性,长度主要在21nt~22nt。靶基因的功能分析表明这些靶基因参与了广泛的生物学功能,尤其是在寄主-病原物互作中占的比重最大。选取25个参与寄主-病原互作的抗性相关基因进行定量验证,发现接种BYDV-GAV后有15个明显下调,6个上调,4个微弱下调,表明测序结果和靶基因预测可靠。推测BYDV-GAV可以通过vsiRNAs干扰寄主抗性基因表达和信号转导,从而实现对感病寄主的侵染。研究结果对揭示BYDV-GAV与小麦互作的分子机制具有重要意义。     

感染大麦黄矮病毒的小麦中vsiRNA特征分析

 RNA沉默是植物中一种保守的抗病毒机制,其以大量病毒来源的小干扰RNA(virus-derived small interfering RNAs,vsiRNAs)的产生为标志,病毒在侵染寄主过程中可通过vsiRNAs靶向寄主转录本以对抗这种防御机制。BYDV-GAV引起的小麦黄矮病导致小麦黄化和矮化症状,对小麦生产构成严重威胁。通过深度测序技术,分析了感染BYDV-GAV的感病小麦品种‘小偃6号’中vsiRNAs的特征,共得到11 384个vsiRNAs,并预测到37 784个寄主靶基因。发现来源于BYDV-GAV基因组的正义和反义链的vsiRNAs的数量分布大致相等,在5’末端具有A和C偏好性,长度主要在21nt~22nt。靶基因的功能分析表明这些靶基因参与了广泛的生物学功能,尤其是在寄主-病原物互作中占的比重最大。选取25个参与寄主-病原互作的抗性相关基因进行定量验证,发现接种BYDV-GAV后有15个明显下调,6个上调,4个微弱下调,表明测序结果和靶基因预测可靠。推测BYDV-GAV可以通过vsiRNAs干扰寄主抗性基因表达和信号转导,从而实现对感病寄主的侵染。研究结果对揭示BYDV-GAV与小麦互作的分子机制具有重要意义。     

Aphid Abundance on Cereals in Autumn Predicts Yield Losses Caused by Barley yellow dwarf virus

ABSTRACT Barley yellow dwarf virus (BYDV) damage to winter cereals and population dynamics of the aphid Rhopalosiphum padi during fall were monitored in fields during 10 years at various locations in the northern half of France. Logistic regression was used to examine whether a simple risk probability algorithm based only on the autumnal population dynamics of R. padi can accurately predict yield losses caused by BYDV and, therefore, the need for insecticide treatment. Results showed that the area under the curve of the percentage of plants infested by R. padi during autumn was highly significantly related to BYDV yield losses. Then, a cost/benefit analysis was performed to estimate the optimal decision threshold resulting in the lowest annual average costs of BYDV damage and control. A "model use" strategy allowed a reduction in the annual average costs of BYDV disease and control of up to 36% when compared with a "prophylactic spraying" strategy. The optimal decision threshold was highly sensitive to variation in disease prevalence. This property was used to propose an easy way to adapt the model to any production situation through the determination of the most accurate decision threshold.     

Development of a High-Throughput Method for Quantification of Plasmopara viticola DNA in Grapevine Leaves by Means of Quantitative Real-Time Polymerase Chain Reaction

ABSTRACT Plasmopara viticola is a strictly biotrophic oomycete that causes downy mildew, which is one of the most important grapevine diseases. Control of the disease is most often achieved by fungicide applications, which may have severe environmental consequences. Therefore, alternative control strategies based on biocontrol agents (BCAs) are currently in development. Thousands of potential BCAs have to be screened for their antagonist efficacy against Plasmopara viticola. Evaluation of their effect on the pathogen can be achieved by detecting the amount of P. viticola DNA in leaves treated with potential antagonists and infected with the pathogen. In this study, a rapid high-throughput method was developed for relative quantification of P. viticola DNA directly from Vitis vinifera leaves by means of multiplex real-time quantitative polymerase chain reaction (PCR) with TaqMan chemistry. This method allows simultaneous amplification, but independent detection, of pathogen and host DNA by using species-specific primers and TaqMan probes that are labeled with different fluorescent dyes. Including detection of V. vinifera DNA in the tests is fundamental because it provides an endogenous reference and allows normalization for variations caused by sample-to-sample differences in DNA extraction, PCR efficiencies, and pipetting volumes. The developed method allows highly sensitive and specific detection of P. viticola DNA (minimal detectable quantity of 0.1 pg). Moreover, high precision and reproducibility of TaqMan assays were observed over a linear range of four orders of magnitude, confirming the reliability of the developed PCR assay. Potential applications range from screening for BCA efficiency to evaluation of fungicide efficacy, or assessment of host resistance.     

Quantification of Fusarium solani f. sp. phaseoli in Mycorrhizal Bean Plants and Surrounding Mycorrhizosphere Soil Using Real-Time Polymerase Chain Reaction and Direct Isolations on Selective Media

ABSTRACT The capacity of the arbuscular mycorrhizal fungus Glomus intraradices in reducing the presence of Fusarium solani f. sp. phaseoli in bean plants and the surrounding mycorrhizosphere soil was evaluated in a compartmentalized experimental system. Quantification of the pathogen and the symbiont in plant tissues, the soil regions of the mycorrhizosphere (rhizosphere and mycosphere), and the bulk soil was accomplished using specific polymerase chain reaction (PCR) primers in real-time PCR assays, culture-dependant methods, and microscopic determination techniques. Nonmycorrhizal bean plants infected with the pathogen had distinctive Fusarium root rot symptoms, while infected plants previously colonized by G. intraradices remained healthy. The amount of F. solani f. sp. phaseoli genomic DNA was significantly reduced in mycorrhizal bean plants and in each mycorrhizosphere soil compartment. The presence of G. intraradices in the mycorrhizosphere was not significantly modified, although the mycorrhizal colonization of roots was slightly increased in the presence of the pathogen. The results suggest that the reduced presence of Fusarium as well as root rot symptoms are caused by biotic and/or abiotic modifications of the mycorrhizosphere as a result of colonization with G. intraradices.     

Plant community diversity influences vector behaviour and Barley yellow dwarf virus population structure

The species composition of a plant community can affect the distribution and abundance of other organisms including plant pathogens. The goal of this study was to understand the role of host diversity in the transmission of two Barley yellow dwarf virus (BYDV) species that share insect vectors and hosts. Greenhouse experiments measured the transmission rate of BYDV species PAV and PAS from infected oat plants to healthy agricultural and wild grasses and from these species back to healthy oat seedlings. In the field component of the study, the rate of spread of PAV and PAS was measured in monoculture plots planted with agricultural grasses. In greenhouse experiments, the aphid vector more readily transmitted PAV from agricultural grasses and more readily inoculated PAS to the wild grass species assayed. In the field experiment, disease prevalence was greater in wheat, but there was no difference in the rate of spread of PAV and PAS. These results indicate an interaction between vector and host genotype that selects for greater PAV transmission in grain crops, contributes to differences in disease prevalence between grass types, and maintains pathogen diversity within the larger plant community (i.e. agricultural and non‐agricultural hosts).