Multiple DNA Markers for Identification of Xanthomonas arboricola pv. juglandis Isolates and its Direct Detection in Plant Samples

Xanthomonas arboricola pv. juglandis (Xaj) is the etiological agent of walnut (Juglans regia L.) bacterial blight (WBB), and has been associated to other walnut emerging diseases, namely brown apical necrosis (BAN) and vertical oozing canker (VOC), altogether severely affecting the walnut production worldwide. Despite the research efforts carried out to disclose Xaj genetic diversity, reliable molecular methods for rapid identification of Xaj isolates and culture-independent detection of Xaj in infected plant samples are still missing. In this work, we propose nine novel specific DNA markers (XAJ1 to XAJ9) selected by dedicated in silico approaches to identify Xaj isolates and detect these bacteria in infected plant material. To confirm the efficacy and specificity of these markers, dot blot hybridization was carried out across a large set of xanthomonads. This analysis, which confirmed the pathovar specificity of these markers, allowed to identify four broad-range markers (XAJ1, XAJ4, XAJ6, and XAJ8) and five narrow-range markers (XAJ2, XAJ3, XAJ5, XAJ7, and XAJ9), originating 12 hybridization patterns (HP1 to HP12). No evident relatedness was observed between these hybridization patterns and the geographic origin from which the isolates were obtained. Interestingly, four isolates that clustered together according the gyrB phylogenetic analysis (CPBF 1507, 1508, 1514, and 1522) presented the same hybridization pattern (HP11), suggesting that these nine markers might be informative to rapidly discriminate and identify different Xaj lineages. Taking into account that a culture-independent detection of Xaj in plant material has never been described, a multiplex PCR was optimized using markers XAJ1, XAJ6, and XAJ8. This triplex PCR, besides confirming the dot blot data for each of the 52 Xaj, was able to detect Xaj in field infected walnut leaves and fruits. Altogether, these nine Xaj-specific markers allow conciliating the specificity of DNA-detection assays with typing resolution, contributing to rapid detection and identification of potential emergent and acutely virulent Xaj genotypes, infer their distribution, disclose the presence of this phytopathogen on potential alternative host species and improve phytosanitary control.

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Genetic Analysis of Phytophthora nicotianae Populations from Different Hosts Using Microsatellite Markers

Phytopathology ◽

10.1094/phyto-11-15-0299-r ◽

2016 ◽

Vol 106 (9) ◽

pp. 1006-1014 ◽

Cited By ~ 15

Author(s):

Antonio Biasi ◽

Frank N. Martin ◽

Santa O. Cacciola ◽

Gaetano Magnano di San Lio ◽

Niklaus J. Grünwald ◽

...

Keyword(s):

Plant Material ◽

Genetic Recombination ◽

Strong Association ◽

Infected Plant ◽

Geographic Origin ◽

Phytophthora Nicotianae ◽

Host Genus ◽

Heterothallic Species ◽

Infected Plant Material ◽

Ornamental Species

In all, 231 isolates of Phytophthora nicotianae representing 14 populations from different host genera, including agricultural crops (Citrus, Nicotiana, and Lycopersicon), potted ornamental species in nurseries (Lavandula, Convolvulus, Myrtus, Correa, and Ruta), and other plant genera were characterized using simple-sequence repeat markers. In total, 99 multilocus genotypes (MLG) were identified, revealing a strong association between genetic grouping and host of recovery, with most MLG being associated with a single host genus. Significant differences in the structure of populations were revealed but clonality prevailed in all populations. Isolates from Citrus were found to be genetically related regardless of their geographic origin and were characterized by high genetic uniformity and high inbreeding coefficients. Higher variability was observed for other populations and a significant geographical structuring was determined for isolates from Nicotiana. Detected differences were related to the propagation and cultivation systems of different crops. Isolates obtained from Citrus spp. are more likely to be distributed worldwide with infected plant material whereas Nicotiana and Lycopersicon spp. are propagated by seed, which would not contribute to the spread of the pathogen and result in a greater chance for geographic isolation of lineages. With regard to ornamental species in nurseries, the high genetic variation is likely the result of the admixture of diverse pathogen genotypes through the trade of infected plant material from various geographic origins, the presence of several hosts in the same nursery, and genetic recombination through sexual reproduction of this heterothallic species.

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Comparison of Xanthomonas arboricola pv. juglandis isolates from walnut trees grown in Romania and Hungary

International Journal of Horticultural Science ◽

10.31421/ijhs/20/1-2/1119 ◽

2014 ◽

Vol 20 (1-2) ◽

Author(s):

A. Bandi

Keyword(s):

Juglans Regia ◽

Infected Plant ◽

Hypersensitive Reaction ◽

Carbohydrate Utilization ◽

Yellow Colour ◽

Nicotiana Tabacum L ◽

Xanthomonas Arboricola ◽

High Degree ◽

Hydrolysis Of

Among diseases that affect walnut, bacterial blight is considered the most important one in all walnut growing areas both from Hungary and Romania. For the determination of susceptibility/resistance of walnut cultivars in our posterior work planned, 61 bacterial isolates were collected from walnuts showing symptoms of blight, with the purpose of isolating Xanthomonas arboricola pv. juglandis (Xaj). The characteristic Xaj colonies, frequently present as saprophytes in the infected plant tissues, were separated from other bacteria according to their morphology, yellow colour, hydrolysis of starch, and oxidation of glucose. All isolates were tested for pathogenicity by hypersensitive reaction on tobacco leaves (Nicotiana tabacum L.), bean pods (Phaseolus vulgaris L) and unripe nuts (Juglans regia L.). Determination of taxonomy of the selected isolates denotes a possible subdivision (races, biotypes) of Xaj occurring in different geographical areas, API 20NE and API 50CH kits were used. Hungarian and Romanian strains showed a high degree of similarity of carbohydrate utilization but slightly differed from the type strain. All were Cu-sensitive.

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Assessment of Xanthomonas arboricola pv. juglandis Bacterial Load in Infected Walnut Fruits by Quantitative PCR

Plant Disease ◽

10.1094/pdis-12-18-2253-re ◽

2019 ◽

Vol 103 (10) ◽

pp. 2577-2586

Author(s):

Leonor Martins ◽

Camila Fernandes ◽

Pedro Albuquerque ◽

Fernando Tavares

Keyword(s):

Quantitative Pcr ◽

Juglans Regia ◽

Bacterial Load ◽

Etiologic Agent ◽

Economic Losses ◽

Rapid Diagnostics ◽

In Planta ◽

Chromosomal Dna ◽

Fruit Samples ◽

Xanthomonas Arboricola

Xanthomonas arboricola pv. juglandis is the etiologic agent of important walnut (Juglans regia L.) diseases, causing severe fruit drop and high economic losses in walnut production regions. Rapid diagnostics and knowledge of bacterial virulence fitness are key to hinder disease progression and apply timely phytosanitary measures. This work describes an X. arboricola pv. juglandis-specific real-time quantitative PCR (qPCR) using X. arboricola pv. juglandis-specific DNA markers to quantify the bacterial load in infected walnut plant tissues. Method validation was achieved using calibration curves obtained with serial dilutions of X. arboricola pv. juglandis chromosomal DNA and standard curves obtained from walnut samples spiked with X. arboricola pv. juglandis cells. High correlations (R2 > 0.990 and > 0.995) and low limits of detection (35 chromosomes/qPCR reaction and 2.7 CFU/qPCR reaction) were obtained for both markers considering the calibration and standard curves, respectively. Assessment of qPCR repeatability, reproducibility, and specificity allowed us to demonstrate the reliability and consistency of the method. Furthermore, in planta quantification of X. arboricola pv. juglandis bacterial load using infected walnut fruit samples showed a higher detection resolution compared with standard PCR detection. By allowing quantification of virulence fitness of distinct X. arboricola pv. juglandis strains in planta, the proposed qPCR method may contribute to assertive risk assessment of walnut diseases caused by X. arboricola pv. juglandis and ultimately help to improve phytosanitary practices.

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Detection of Peach latent mosaic viroid by PCR

Plant Protection Science ◽

10.17221/10458-pps ◽

2017 ◽

Vol 38 (SI 2 - 6th Conf EFPP 2002) ◽

pp. 249-251

Author(s):

P. Ryšánek ◽

M. Zouhar ◽

M. Hassan

Keyword(s):

Czech Republic ◽

Plant Material ◽

Rna Extraction ◽

Infected Plant ◽

Potato Spindle Tuber Viroid ◽

Rna Sequences ◽

The Czech Republic ◽

The World ◽

Infected Plant Material ◽

Sensitive Method

Peach latent mosaic viroid (PLMVd) is widespread in peach all over the world. It has never been reported from the Czech Republic. That is why we adapted specific and sensitive method for its detection, PCR, to be able to prove its possible occurrence and for certification purposes. Primers PLMVdR, PLMVdF1 and PLMVdF2 were designed on the basis of published RNA sequences. Products of amplification are 208 and 114 bp long for PLMVdF1 and PLMVdF2, respectively. Four PLMVd isolates from Dr Di Serio (CNR Bari) were used as standards. Potato spindle tuber viroid and Hop latent viroid infected plant material and also healthy material were used to check detection specifity. Both RNA extraction from plant material and PCR were optimalized so that this method of PLMVd detection can also be used for certification purposes.

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Multiplex PCR for specific and robust detection of Xanthomonas campestris pv. musacearum in pure culture and infected plant material

Plant Pathology ◽

10.1111/j.1365-3059.2011.02534.x ◽

2011 ◽

Vol 61 (3) ◽

pp. 489-497 ◽

Cited By ~ 22

Author(s):

J. Adriko ◽

V. Aritua ◽

C. N. Mortensen ◽

W. K. Tushemereirwe ◽

J. Kubiriba ◽

...

Keyword(s):

Multiplex Pcr ◽

Pure Culture ◽

Plant Material ◽

Xanthomonas Campestris ◽

Infected Plant ◽

Robust Detection ◽

Infected Plant Material

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Analysis by PCR-based markers using designed primers to study relationships between species of Hordeum (Poaceae)

Genome ◽

10.1139/g98-109 ◽

1999 ◽

Vol 42 (1) ◽

pp. 129-138 ◽

Cited By ~ 6

Author(s):

Alfredo De Bustos ◽

Consuelo Soler ◽

Nicolás Jouve

Keyword(s):

Dna Markers ◽

Genomic Dna ◽

Plant Material ◽

Dna Analysis ◽

Balearic Islands ◽

Hordeum Bulbosum ◽

Perennial Species ◽

Structural Genes ◽

Genomic Libraries ◽

Cultivated Species

An investigation was made into the relationships between six species or subspecies of the genus Hordeum that grow in the Iberian Peninsula and Balearic Islands. Plant material included samples of 19 populations of the annual species H. marinum subsp. marinum, H. marinum subsp. gussoneanum, H. murinum subsp. murinum, and H. murinum subsp. leporinum, and the perennial species H. bulbosum and H. secalinum. Relationships were analysed using PCR-based molecular markers. Thirteen sets of primers were designed and synthesized based on sequences of mapped RFLPs from genomic libraries and conserved regions of structural genes of known function in cultivated species. Primers were used to amplify genomic DNA from all populations. The number of amplified products ranged from 1 to 18 per primer and a total of 168 markers were scored. The markers revealed different degrees of relationships. Hordeum bulbosum was clearly separated from the rest. The populations of both subspecies of H. murinum were closely related. H. marinum subsp. gussoneanum appeared to be closely related to H. secalinum, yet relatively separated from its conspecific subspecies marinum. The use of a large number of DNA markers in this kind of analysis is discussed.Key words: Hordeum, barley, DNA markers, restriction endonucleases, variability, DNA analysis.

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Overwintering Diseased Plant Parts and Newly Infected Flowers and Fruit as Sources of Inoculum for Colletotrichum acutatum in Sour Cherry

Plant Disease ◽

10.1094/pdis-11-16-1599-re ◽

2017 ◽

Vol 101 (7) ◽

pp. 1207-1213 ◽

Cited By ~ 4

Author(s):

Arne Stensvand ◽

Jorunn Børve ◽

Venche Talgø

Keyword(s):

Plant Material ◽

Infected Plant ◽

Sour Cherry ◽

Growing Season ◽

Early Spring ◽

Tissue Type ◽

Colletotrichum Acutatum ◽

Diseased Plant ◽

Plant Parts ◽

Southern Norway

Production of inoculum of Colletotrichum acutatum from both previously infected and overwintered tissue, as well as newly developed plant tissue of sour cherry (Prunus cerasus), was studied in southern Norway. Plant parts were sampled from commercial, private, or research orchards, and incubated for 2 to 14 days (time depended on tissue type) in saturated air at 20°C. In early spring, abundant sporulation was found on scales of overwintered buds and shoots. A mean of 35% infected buds in four cultivars was observed, with a maximum of 72% of the buds infected in one of the samples. Over 3 years, the seasonal production of overwintered fruit and peduncles of cv. Fanal infected the previous year was investigated. In all three years, the infected plant material was placed in the trees throughout the winter and the following growing season; in two of the years, fruit and peduncles were also placed on the ground in the autumn or the following spring. Old fruit and peduncles formed conidia throughout the season, with a peak in May and June. Spore numbers declined over the season, but the decline was more rapid for plant material on the ground than in the trees. On average over 2 years, 68.7, 24.0, or 7.3% of the inoculum came from fruit placed in the trees, placed on the ground in spring, or placed on the ground the preceding autumn, respectively. The number of fruit and peduncles attached to the trees in a planting of cv. Hardangerkirsebær was followed from February to July one year, and although there was a decline over time, fruit and/or their peduncles were still attached in substantial numbers in July, thus illustrating their potential as sources of inoculum. In observations over 2 years in a heavily infected orchard of cv. Stevnsbær, 75 and 47% of flowers and newly emerged fruit, respectively, were infected. Artificially inoculated flowers and fruit produced conidia until harvest, with a peak in mid-July. It may be concluded that previously infected and overwintered, as well as newly emerged tissue of sour cherry, may serve as sources of inoculum of C. acutatum throughout the growing season.

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First Report of Walnut Canker Caused by Fusarium incarnatum from India

Plant Disease ◽

10.1094/pdis-04-11-0352 ◽

2011 ◽

Vol 95 (12) ◽

pp. 1587-1587

Author(s):

B. Singh ◽

C. S. Kalha ◽

V. K. Razdan ◽

V. S. Verma

Keyword(s):

Fusarium Species ◽

Juglans Regia ◽

Infected Plant ◽

Distilled Water ◽

Conidial Suspension ◽

State University ◽

Single Spore ◽

Jammu And Kashmir ◽

First Report ◽

Branch Dieback

While screening newly introduced cultivars of walnut (Juglans regia) at Bhaderwah (Mini Kashmir), Jammu and Kashmir, India in September 2008, 60% of grafted plants were found to be dying because of a cankerous growth observed on seedling stems. Later, these symptoms extended to lateral branches. In the surveyed nurseries, cvs. SKU 0002 and Opex Dachaubaria were severely affected by the disease. Cankers were also observed in all walnut nurseries in the area with several wild seedlings also being observed to be exhibiting similar cankerous symptoms on stem and branches. Necrotic lesions from cankerous tissues on seedling stems were surface disinfested with 0.4% NaOCl for 1 min and these disinfected cankerous tissues were grown on potato dextrose agar (potato-250 g, dextrose-15 g, agar-15 g, distilled water-1 liter). A Fusarium sp. was isolated consistently from these cankerous tissues, which was purified using single-spore culture. Carnation leaf agar was used for further culture identification (2,3). The fungal colony was floccose, powdery white to rosy in appearance when kept for 7 days at 25 ± 2°C. Macroconidia were straight to slightly curved, four to eight septate and 30 to 35 × 3.5 to 5.7 μm. These are characteristics consistent with Fusarium incarnatum (3). Pathogenicity was confirmed by spraying a conidial suspension (1 × 106 conidia/ml) onto bruised branches of 1-year-old walnut plants (cv. Opex Dachaubaria) while sterile distilled water sprays were used for the controls. Inoculated plants were incubated at 20 ± 2°C and 85% relative humidity for 48 h. Fifty days following inoculation, branch dieback followed by canker symptoms developed on inoculated plants. Control plants remained healthy with no symptoms of canker. F. incarnatum (Roberge) Sacc. was repeatedly isolated from inoculated walnut plants, thus satisfying Koch's postulates. Infected plant material has been deposited at Herbarium Crytogamae Indiae Orientalis (ITCC-6874-07), New Delhi. To our knowledge, this is the first report of walnut canker caused by F. incarnatum (Roberge) Sacc. from India. This fungus was previously reported to be affecting walnut in Italy (1) and Argentina (4). References: (1) A. Belisario et al. Informatore Agrario 21:51, 1999. (2) J. C. Gilman. A Manual of Soil Fungi. The Iowa State University Press, Ames, 1959. (3) P. E. Nelson et al. Fusarium Species. An Illustrated Manual for Identification. The Pennsylvania State University Press, University Park, 1983. (4) S. Seta et al. Plant Pathol. 53:248, 2004.

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Genetic diversity of pomegranate germplasm collection from Spain determined by fruit, seed, leaf and flower characteristics

10.7287/peerj.preprints.2048 ◽

2016 ◽

Author(s):

Juan José Martínez ◽

Pablo Melgarejo ◽

Pilar Legua ◽

Francisco García ◽

Francisca Hernández

Keyword(s):

Genetic Diversity ◽

Cluster Analysis ◽

Plant Material ◽

Phenotypic Variability ◽

Geographical Area ◽

Geographic Origin ◽

Germplasm Management ◽

Morphometric Characteristics ◽

Germplasm Bank ◽

Improvement Programs

Background . Miguel Hernandez University ( Spain ) created a germplasm bank of the varieties of pomegranate from different Southeastern Spain localities in order to preserve the crop’s wide genetic diversity. Once this collection was established, the next step was to characterize the phenotype of these varieties to determine the phenotypic variability that existed among all the different pomegranate genotypes, and to understand the degree of polymorphism of the morphometric characteristics among varieties. Methods. Fifty-three pomegranate (Punica granatum L.) accessions were studied in order to determine their degree of polymorphism and to detect similarities in their genotypes. Thirty-one morphometric characteristics were measured in fruits, arils, seeds, leaves and flowers, as well as juice characteristics including content, pH, titratable acidity, total soluble solids and maturity index. ANOVA, principal component analysis, and cluster analysis showed that there was a considerable phenotypic diversity (and presumably genetic). Results. The cluster analysis produced a dendrogram with four main clusters. The dissimilarity level ranged from 1 to 25, indicating that there were varieties that were either very similar or very different from each other, with varieties from the same geographical areas being more closely related. Within each varietal group, different degrees of similarity were found, although there were no accessions that were identical. These results highlight the crop’s great genetic diversity, which can be explained not only by their different geographical origins, but also to the fact that these are native plants that have not come from genetic improvement programs. The geographic origin could be, in the cases where no exchanges of plant material took place, a key criterion for cultivar clustering. Conclusions. As a result of the present study, we can conclude that among all the parameters analyzed, those related to fruit and seed size as well as the juice’s acidity and pH had the highest power of discrimination, and were, therefore, the most useful for genetic characterization of this pomegranate germplasm banks. This is opposed to leaf and flower characteristics, which had a low power of discrimination. This germplasm bank, more specifically, was characterized by its considerable phenotypic (and presumably genetic) diversity among pomegranate accessions, with a greater proximity existing among the varieties from the same geographical area, suggesting that over time, there had not been an exchange of plant material among the different cultivation areas. In summary, knowledge on the extent of the genetic diversity of the collection is essential for germplasm management. In this study, these data may help in developing strategies for pomegranate germplasm management and may allow for more efficient use of this germplasm in future breeding programs for this species.

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Vascular Streak Dieback: Penyakit Baru Tanaman Kakao di Sumatera Barat

Jurnal Fitopatologi Indonesia ◽

10.14692/jfi.12.4.142 ◽

2016 ◽

Vol 12 (4) ◽

pp. 142

Author(s):

Jumsu Trisno ◽

Reflin Reflin ◽

Martinius Martinius

Keyword(s):

Agar Medium ◽

Disease Incidence ◽

Infected Plant ◽

Potato Dextrose Agar ◽

Fungal Isolation ◽

Potato Dextrose Agar Medium ◽

First Report ◽

Plant Samples ◽

Dead Plant

Vascular streak dieback (VSD) symptoms was reported recently in several cacao plantations in West Sumatera. Disease incidence reached 58.82–100% with disease intensity of 24.29–44.71%. In some cases, dead plant was also found. Fungal isolation was performed to identify the agents associated with VSD. Plant samples showing VSD symptoms was collected from 3 locations of cacao production center in West Sumatera, i.e. Limapuluh Kota regency, Padang Pariaman regency, and Padang city. Small pieces of leaf and twig were plated on water agar and potato dextrose agar medium for fungal isolation. Morphology of hifa, basidiocarp, and basidiospora observed from fungi colonies indicated the presence of Ceratobasidium theobromae on infected plant samples. This is the first report on the association of C. theobromae on cacao in West Sumatera.

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