Improved PCR-based Assays for Pre-symptomatic Diagnosis of Light Leaf Spot and Determination of Mating Type of Pyrenopeziza Brassicae on Winter Oilseed Rape

An existing PCR-based method for diagnosis of the winter oilseed rape (Brassica napus ssp oleifera) fungal pathogen Pyrenopeziza brassicae (cause of light leaf spot) was improved by the development of a pair of primers (PbN1 and PbN2) for use in nested-PCR reactions. The nested-PCR technique improved the detection of P. brassicae DNA in vitro by three orders of magnitude over that achieved using the first-round PCR primers (Pb1 and Pb2). In controlled environment experiments, the nested-PCR assay detected P. brassicae within infected B. napus leaves before visible light leaf spot symptoms developed and earlier than was possible by incubating infected leaves in polyethylene bags to promote sporulation of P. brassicae. A three-primer PCR technique using the primers PbM-1-3, PbM-2 and Mt3 was developed to distinguish between the two mating types (MAT-1 and MAT-2) of P. brassicae. This technique was able to determine the mating types present within DNA extracted from infected plant tissue, including tissue infected with both mating types together.     

Inoculum and disease dynamics of circular leaf spot of persimmon caused by Mycosphaerella nawae under semi-arid conditions

The epidemiology of circular leaf spot of persimmon, caused by Mycosphaerella nawae, was studied in a semi-arid area in Spain for two consecutive years. No conidia were observed on diseased leaves and all infections were thought to be caused by ascospores formed in the leaf litter. Ascospores were released mainly in April and May, but relatively low numbers in June were able to induce severe symptoms on trap plants. Temperature was not significantly correlated with ascospore catches or disease incidence on trap plants, indicating that it was not a limiting factor for disease development during the period of study. Rainfall was above normal, but still considerably lower than in endemic areas of Korea. Most infections coincided with rains, but the disease was observed also on trap plants exposed to less than 1?mm of precipitation and even in the absence of rain. Orchards were flood irrigated once inoculum deposits in the leaf litter had already been depleted, so it was not possible to determine its effects on ascospore release and disease development. The use of a wind tunnel to determine inoculum production allowed detection of physiologically mature ascospores of M. nawae in the leaf litter 1?C2?weeks before they were released to air in the orchard. Disease progress was fitted to the monomolecular growth curve, associated with monocyclic pathogens and diseases with a variable incubation period as a function of the host phenology.     

Overwintering of brown leaf spot fungus, Mycochaetophora gentianae, in infected gentian leaves as the primary inoculum source

Overwintering of the brown leaf spot fungus, Mycochaetophora gentianae, in infected gentian leaves was studied in Iwate, northern Japan. Sporophores were produced on overwintered, infected leaves when they were sampled from January to July, but not in August after incubation in high humidity at 15 °C. Symptoms developed on gentian plants grown in soil artificially infested with overwintered, infected leaves that were either left throughout the experiments or removed before planting. Few lesions developed when plants were grown in soil infested with conidia. These results indicate that M. gentianae can overwinter in infected leaves, which act as the primary inoculum source.     

Molecular identification of a Candidatus phytoplasma (Group16SrV-D) coding partial uvrB gene and degV gene on a new host – mesta (Hibiscus sabdariffa) – with phyllody and reddening of leaves in India

Mesta (Hibiscus sabdariffa) is an important bast fiber crop. In August 2011, there was an outbreak of a phytoplasma-like disease on H. sabdariffa in different villages of the northern coastal mesta-growing region of Andhra Pradesh, India, covering mainly two districts – Srikakulam and Vijayanagaram. The infected plants showed characteristic symptoms such as phyllody and reddening of leaves. PCR with P1/P7 universal primer pair of 16 S rDNA yielded amplicons of 1850 bp from all symptomatic mesta leaf samples similar to samples of brinjal little leaf (phytoplasma positive reference control). However, asymptomatic samples were not amplified. Multiplex nested-PCR showed simultaneous amplification of DNA fragments with phytoplasma specific primers, viz., P1/P7 universal primer pair of 16 S rDNA, nested primer pair R16F2n/R2, uvrB and DegV gene-specific uvrB-degVF/R primer generating amplicons of 1850 bp, 1200 bp and 1023bp, respectively. However, SecY-map gene specific primer SecY-mapF/R was not amplified. The 1023 bp nucleotide sequence of uvrB and DegV gene of the phytoplasma was deposited in the GenBank (NCBI) with the accession no. JX975061. NCBI BLASTn analysis of the 1023 bp products showed that the phytoplasma strain belonged to elm yellows group (16SrV-D). This is the first report that Hibiscus sabdariffa is infected by a phytoplasma and we named it mesta phyllody disease (MPD).     

Quantitative detection of <Emphasis Type="Italic">Monosporascus cannonballus</Emphasis> in infected melon roots using real-time PCR

A method was developed for the specific detection, identification and quantification of Monosporascus cannonballus in infected melon roots based on real-time PCR (SYBR® Green chemistry) targeting the ITS1 region of the rDNA conserved between different strains of the pathogen. The specificity of the reaction was assessed using a number of fungi taxonomically and ecologically related to M. cannonballus. The method was highly sensitive and M. cannonballus was first detected in the roots of a susceptible Piel de Sapo cultivar 2 days after inoculation, before symptom appearance. Although conventional PCR methods could also provide such a specific and sensitive detection, real-time PCR was also able to produce reliable quantitative data over a range of 4 orders of magnitude (from 5 ng to 0.3 pg). The method allowed the quantitative monitoring of fungal growth from the very first stages of infection, and was successfully employed in the early screening of resistance. The assessment of disease progress and severity obtained with real-time PCR was more accurate than that obtained with the visual scoring of root lesions or root biomass losses. Therefore, there exists a great potential for its implementation in those steps of breeding programmes where high accuracy is required.     

Fast detection by loop-mediated isothermal amplification (LAMP) of the three begomovirus species infecting tomato in Panama

Potato yellow mosaic Panama virus (PYMPV), Tomato leaf curl Sinaloa virus (ToLCSiV) and Tomato yellow mottle virus (TYMoV) of genus Begomovirus (family Geminiviridae) are the only three begomovirus species detected infecting tomato (Solanum lycopersicum L.) in Panama. PYMPV, ToLCSiV and TYMoV induce symptoms of stunting, yellowing, curling, distortion of leaves and reduction of fruit size and cause important economic loses. A loop-mediated amplification under isothermal conditions (LAMP) assay was developed for the individual detection of these three begomovirus species by using a set of three primer pairs specific per each one of them. Amplification products were visualized by gel electrophoresis or direct Gel-Red staining of DNA into the reaction tube. PYMPV, ToLCSiV and TYMoV were detected in total DNA extracts obtained from different plant tissues such as leaves, stems, flowers, fruits and roots of infected tomato plants collected in different production regions of Panama. LAMP sensitivity was similar to that of conventional PCR but, the first procedure was faster and cheaper than the last one. Moreover, all three viruses were successfully detected by LAMP and not by conventional PCR from sap extracts obtained from leaf tissues of infected tomato plants which were embedded into 3MM Whatman paper and stored several days, facilitating the samples processing as well as the material movement among different laboratories. Therefore, LAMP is a specific, rapid and cheap procedure to detect all three begomoviruses infecting tomato in Panama and it is suitable for field surveys and sanitation programs.     

Assessment of the loose smut fungi (Ustilago nuda and U. tritici) in tissues of barley and wheat by fluorescence microscopy and real-time PCR

Loose smut fungi of barley and wheat (Ustilago nuda and U. tritici, respectively) colonize the plant without causing obvious disease symptoms before heading. The availability of diagnostic methods to detect and follow the growth of these pathogens in the plant would therefore be highly advantageous for both resistance breeding and the development of effective seed treatments. Using seed lots of barley and wheat highly infected with loose smut, we studied the early establishment of the loose smut pathogens in the plant by fluorescence microscopy. In hand-cut sections stained with the fluorochrome Blankophor?, fungal hyphae were observed to invade the shoot apical meristem and leaf primordia during the first days after the onset of germination. At the first node stage the ear and leaf primordia were generally extensively colonized. Hyphae of U. nuda were also regularly observed in high density in the nodes. A protocol was developed for the specific amplification of U. nuda and U. tritici DNA extracted from infected plant tissue. PCR screening of U nuda in seedlings from infected and healthy seed lots was compared to ELISA, microscopy and ultimately head infection of mature plants derived from tillers of the tested seedlings. The results indicated that a prediction of loose smut infection by real-time PCR is possible at the second leaf stage, and that the assay is equally suited for use with spring and winter varieties of barley and wheat.     

Contribution of <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> toxins to avocado white root rot

By transversely cutting infected avocado plant stems and using PCR techniques on avocado leaves, two experiments were carried out to determine whether Rosellinia necatrix can invade avocado vascular tissues. We were unable to detect the pathogen in either stems or leaves in either experiment, so we concluded that R. necatrix does not invade the vascular system of the plant. Additionally, the toxins produced by the pathogen were also studied to determine whether such toxins could contribute to the wilting and death of avocado plants infected by R. necatrix, having an effect on avocado leaves, where they can hinder the photosynthetic process. First, we isolated and identified the toxins cytochalasin E and rosnecatrone from filtrates of six R. necatrix isolates. Second, we tried to detect cytochalasin E in sap and leaves from infected avocado plants, and it was not detected at the minimum level of 50 μg/kg in leaves or 25 μg/kg on sap. Finally, we observed changes in fluorescence emitted by the avocado leaf surface (to detect photosynthetic efficiency) after inoculating avocado plants with this toxin. Fluorescence was higher in the leaves of plants immersed in toxin solution after 4 and 8 days, but not after longer periods of time. In this work, we demonstrated that although R. necatrix is not a fungus that invades the vascular system, its toxins are probably involved in the wilting and death of infected avocado plants, decreasing the efficiency of photosynthesis.     

Early detection of sugar beet pathogen Ramularia beticola in leaf and air samples using qPCR

A quantitative PCR method (qPCR) was developed for the detection and quantification of Ramularia beticola causing Ramularia leaf spot in sugar beet. R. beticola specific primers were designed based on the internal transcribed spacer region 2 (ITS2). The assay was applied on DNA extracted from spores trapped on tape from Burkard spore traps placed in an artificially inoculated sugar beet field trial and in two sugar beet fields with natural infections. R. beticola DNA was detected at variable amounts in the air samples 14 to 16 days prior to first visible symptoms. R. beticola DNA was detected in air samples from fields with natural infection at significant and increasing levels from development of the first symptoms, indicating that spore production within the crop plays a major role in the epidemic development of the disease. Sugar beet leaves sampled from the inoculated field trial were also tested with the qPCR assay. It was possible to detect the presence of R. beticola in the leaves pre-symptomatic at least 10 days before the occurrence of the visible symptoms of Ramularia leaf spot. This is the first report of a molecular assay, which allows screening for the presence of R. beticola in plant material and in air samples prior to the appearance of visible symptoms. An early detection has potential as a tool, which can be part of a warning system predicting the onset of the disease in the sugar beet crop and helping to optimise fungicide application.     

Development and evaluation of specific PCR and LAMP assays for the rapid detection of Phytophthora melonis

Phytophthora melonis is a widespread and devastating pathogen for the Cucurbitaceae family. Early and accurate detection of P. melonis is essential to control the disease in the field. To establish a simple, visual, and rapid detection system for P. melonis, we developed nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) systems based on the Ras-related protein (Ypt1) gene. All 36 isolates of P. melonis, from geographically distinct counties in China, yielded positive detection results on LAMP or nested PCR assays. No cross reaction was observed with other oomycetes or fungal pathogens. A sensitivity assay showed that both methods had a detection limit of 10 fg genomic DNA. We also detected P. melonis in diseased cucumber tissues and soils, and evaluated positive detection rates using LAMP, nested PCR, and conventional isolation methods. The results suggest that the LAMP assay has the greatest potential for active detection of P. melonis in regions that are at risk of contracting the disease, and for use in resource-poor settings.     

Detection of Latent Sphaeropsis sapinea Infections in Austrian Pine Tissues Using Nested-Polymerase Chain Reaction

ABSTRACT Sphaeropsis sapinea is the causal agent of Sphaeropsis tip blight disease of pines. Past surveys of diseased and symptomless Austrian and Scots pines revealed that latent infections of symptomless shoots by S. sapinea are common. The role of these latent infections in the tip blight disease is unknown. A sampling technique and nested-polymerase chain reaction (PCR) protocol were developed to detect latent S. sapinea in symptomless pine shoots. The sampling protocol was designed to be minimally destructive to the shoot so it could be preserved for further studies. The primers that were developed were specific for S. sapinea DNA and did not amplify DNA from any of 13 other endophytic fungal species that were commonly isolated from symptomless pine shoots. The PCR primers also amplified DNA of Botryosphaeria obtusa, which was, however, rare in symptomless Austrian pine tissues. The protocol detected as little as 0.93 pg of S. sapinea DNA in terminal bud samples and 10.4 pg of DNA in bark samples. Correlation (chi-square) analyses indicated that the nested-PCR protocol detected latent S. sapinea infections in both bud and bark samples with an efficiency that was statistically equivalent to isolating the fungus from the tissue. The nested-PCR protocol will make it possible to more quickly identify latent S. sapinea infections in symptomless pine shoots and should be useful in future studies of the latency phenomenon.     

Direct detection of <Emphasis Type="Italic">Taphrina deformans</Emphasis> on peach trees using molecular methods

The ascomycetous fungus Taphrina deformans is the agent of peach leaf curl, a worldwide disease of peach potentially devastating to both crop yields and tree longevity. Conspicuous leaf curl symptoms result from the invasion of host tissue by the strictly parasitic mycelial phase of the T. deformans dimorphic life-cycle. Successful isolation of the fungus in pure culture is cumbersome and limited to late spring/early summer (time of ascospore discharge from infected leaves) and only rarely has the asymptomatic yeast phase been isolated from buds. Molecular methods, namely those based on the hybridisation of nucleic acids, are advantageous for diagnostic purposes since they do not require isolation of the fungus on culture media. Direct amplification using the polymerase chain reaction (PCR) and fluorescent in situ hybridisation (FISH) were tested for diagnosis of peach leaf curl disease in order to provide a fast and reliable method for disease risk assessment. Specific primers and probes were designed based on available ribosomal DNA sequence data. Positive and specific diagnoses of peach leaf curl were achieved with primer TDITS1, using PCR-detection, and probe TDE634, using FISH, both on infected leaves and in washings of asymptomatic peach buds.     

Occurrence of Groundnut ringspot virus on Brazilian peanut crops

A high incidence of plants with mosaic, chlorotic spots, ringspots, necrosis, smaller leaves, and stunting was observed on peanut crops (Arachis hypogaea L.) in Itápolis, São Paulo State, Brazil. Transmission electron microscope examination of thin sections of infected leaves revealed the presence of spheroidal particles, ca. 80 nm in diameter, suggestive of Tospovirus. A DNA fragment of ~600 bp was amplified by RT-PCR from total RNA extracted from infected tissues using primers specific for the nucleocapsid gene of Groundnut ringspot virus (GRSV). Nucleotide and deduced amino acid sequences of the fragments showed high identities with known GRSV isolates.     

柑橘黄龙病nested-PCR检测技术在柑橘苗木生产中的应用

柑橘黄龙病(huanglongbing, HLB)是柑橘生产上的一种毁灭性病害。本研究以柑橘叶脉为材料,采用nested-PCR技术对广西部分柑橘苗圃苗木进行了黄龙病检测。结果表明,nested-PCR技术不仅可以检测已经表现症状的苗木样品,还可以检测出带病但不显症的苗木样品。2007年共检测柑橘苗木样品1 950个,2008年共检测样品1 480个,黄龙病苗木检出率分别为10.31％和6.22％。本研究对及早控制带病苗木的传播,繁育柑橘无病毒苗木具有重要的意义。     

Molecular diagnostics for the sigatoka disease complex of banana

ABSTRACT The Sigatoka disease complex of banana involves three related ascomycetous fungi, Mycosphaerella fijiensis, M. musicola, and M. eumusae. The exact distribution of these three species and their disease epidemiology remain unclear, because their symptoms and life cycles are rather similar. Disease diagnosis in the Mycosphaerella complex of banana is based on the presence of host symptoms and fungal fruiting structures, which hamper preventive management strategies. In the present study, we have developed rapid and robust species-specific molecular-based diagnostic tools for detection and quantification of M. fijiensis, M. musicola, and M. eumusae. Conventional species-specific polymerase chain reaction (PCR) primers were developed based on the actin gene that detected DNA at as little as 100, 1, and 10 pg/mul from M. fijiensis, M. musicola, and M. eumusae, respectively. Furthermore, TaqMan real-time quantitative PCR assays were developed based on the beta-tubulin gene and detected quantities of DNA as low as 1 pg/mul for each Mycosphaerella sp. from pure cultures and DNA at 1.6 pg/mul per milligram of dry leaf tissue for M. fijiensis that was validated using naturally infected banana leaves.     

Validation of a real‐time PCR method for the detection of Phytophthora ramorum1

To validate a real‐time PCR method for the detection of Phytophthora ramorum, an intra‐laboratory procedure was developed. The specificity of the TaqMan probe/primer sets was determined by carrying out real‐time PCR on total DNA extracted from pure culture of several Phytophthora species. The limit of detection and the potential effects of plant substrates were evaluated by conducting the test on total DNA from healthy plant materials (Rhododendron spp., Viburnum spp. and Pieris spp.) spiked with known amounts of P. ramorum genomic DNA. The PCR efficiency was estimated through the linear regression of the dilution curve. Precision of the TaqMan assay was assessed on material from a single artificially infected plant (Rhododendron spp.). Two kinds of tissues were tested: a severely infected twig and an apparently healthy leaf. Intra‐assay repeatability was evaluated on 10 replicates of the same DNA sample analysed in a single assay. Inter‐assay reproducibility was evaluated on the same DNA sample amplified over five separate assays while the intersample reproducibility was evaluated on separate DNA extractions of four samples from both plant tissues amplified in a single assay.     

The Eurotiomycete <Emphasis Type="Italic">Apinisia graminicola</Emphasis> as the causal agent of a leaf spot disease on the energy crop <Emphasis Type="Italic">Miscanthus</Emphasis> x <Emphasis Type="Italic">giganteus</Emphasis> in Northern Germany

Miscanthus x giganteus is a fast growing, perennial energy crop for temperate climates. Because of its high annual biomass production rates and its characteristics as a low-input crop, an expansion of field cultivation can be anticipated to cover increasing demands for sustainable biomass production. However, knowledge about pathogens that could have an impact on biomass production is still limited for M. giganteus. Here, we report about the isolation of the filamentous fungus Apinisia graminicola from necrotic leaf lesions of M. giganteus grown on a field trial plot in Northern Germany. Inoculation assays with the isolated A. graminicola strain confirmed its capacity to cause a leaf spot disease on M. giganteus. Additional inoculation assays revealed that A. graminicola also caused necrotic lesions on leaves of the model grass Brachypodium distachyon. Generally, symptoms of A. graminicola-caused leaf spot disease were stronger on B. distachyon compared to M. giganteus. Incubation temperatures above 22 °C during A. graminicola infection resulted in stronger disease symptoms on both, M. giganteus and B. distachyon leaves. Microscopic analysis of cross sectioned, infected leaf tissue revealed an epiphytic mycelium formation on the surface and an endophytic colonization of the mesophyll leave tissue, especially in M. giganteus. Our results revealed that the isolated A. graminicola strain is a causal agent of a leaf spot disease on grass leaves. Its potential on endophytic growth in M. giganteus might open new possibilities in studying this type of plant-fungal interaction on a cellular and molecular level in an energy crop.     

In planta-polymerase-chain-reaction detection of the wilt-inducing pathotype ofFusarium oxysporumf.sp.cicerisin chickpea (Cicer arietinumL.)

A 1.6 kb fragment of random amplified polymorphic DNA (RAPD-PCR, polymerase chain reaction), which was specific for race 5, a wilt-inducing isolate ofFusarium oxysporumf.sp.ciceris(Foc), was cloned and sequenced. This fragment was not detected in RAPD-PCR reactions with DNA from yellowing-inducing pathotypes ofFoc, or from other fungi tested. Specific PCR primers were designed from the sequence data and used to detect the presence of the fungus in genomic DNA isolated from symptomless chickpea plants, 16 days after inoculation. A single, 1.5 kb PCR product was only observed in PCR reactions with DNA from plants infected with a wilt-inducing isolate. No products were observed in reactions with DNA from plants infected with yellowing-inducing pathotypes, or from DNA isolated from uninfected chickpea cultivar controls. Southern hybridization demonstrated homology between the second PCR product and the original specific wilt-associated RAPD fragment. PCR products were detected with DNA extracted from roots and stem tissue, but no fungal DNA was detected in leaf tissue of the same infected plants. In a blind trial, the specific primers correctly identified the fungal pathotype in four different, wilt-infected chickpea cultivars.     

Quantitative detection of <Emphasis Type="Italic">Colletotrichum godetiae</Emphasis> and <Emphasis Type="Italic">C. acutatum sensu stricto</Emphasis> in the phyllosphere and carposphere of olive during four phenological phases

A duplex qPCR detection method was developed to detect and quantify Colletotrichum godetiae and C. acutatum sensu stricto (s.s.) in olive tissues. The method proved highly specific and sensitive with a detection limit of 10 pg for each pathogen. The analysis of green and senescent leaves, fertilized fruitlets with floral residues, green fruit and symptomatic and asymptomatic fruit collected in May, June, October and December revealed a high incidence of both C. godetiae and C. acutatum s.s. in Calabria, southern Italy. In comparison with previous reports, these results highlighted an ongoing population shift from C. godetiae to C. acutatum s.s. Interestingly, C. godetiae was slightly more abundant in terms of number of infected samples, yet the quantity of C. acutatum in infected samples was always higher, suggesting greater aggressiveness and/or sporulation ability of the latter pathogen. The populations of both C. godetiae and C. acutatum s.s. increased sharply in December even though both pathogens were detected widely in asymptomatic samples in May, June and October, confirming an important role of latent infections in the disease cycle. A large quantity of both C. godetiae (1.7 × 10⁸ cells/mg of tissue) and C. acutatum s.s. (7.5 × 10⁸ cells/mg of tissue) was estimated in symptomatic fruit, presenting an enormous inoculum potential for secondary infections. Two other important observations were a high incidence and quantity of both pathogens in senescent leaves and in fertilized fruitlets with floral residues as compared to green leaves.     

Effects of temperature,water regime and irrigation system on the release of ascospores of Mycosphaerella nawae,causal agent of circular leaf spot of persimmon

Experiments were conducted under controlled conditions to quantify the effects of temperature, water regime and irrigation system on the release of Mycosphaerella nawae ascospores from leaf litter in Spanish persimmon orchards. The effect of temperature on ascospore release was best described by a Gompertz model. The end of the lag phase of ascospore release occurred at 9·75°C, and the end of the exponential phase at 15·75°C. Few ascospores were discharged from dry leaves wetted with 0·1 or 0·5 mm water, but significant amounts were recovered with 1–50 mm water. About half of the total ascospores were released after three wetting and drying cycles, but 32 cycles were necessary for a complete discharge. No significant difference in ascospore release was detected when the leaf litter was wetted by flood and drip irrigation. However, considering the proportion of soil area wetted in both systems, inoculum release was significantly reduced by drip irrigation. The potential of drip irrigation as a cultural control measure should be investigated.