scholarly journals Nanopore sequencing for the detection and identification of Xylella fastidiosa subspecies and sequence types from naturally infected plant material

2021 ◽  
Author(s):  
Luigi Faino ◽  
Valeria Scala ◽  
Alessio Albanese ◽  
Vanessa Modesti ◽  
Alessandro Grottoli ◽  
...  
Keyword(s):  
Plant Material ◽  
Xylella Fastidiosa ◽  
Infected Plant ◽  
Nanopore Sequencing ◽  
Detection And Identification ◽  
Sequence Types ◽  
Infected Plant Material

scholarly journals Nanopore sequencing for the detection and the identification of Xylella fastidiosa subspecies and sequence types from naturally infected plant material

2019 ◽  
Author(s):  
Luigi Faino ◽  
Valeria Scala ◽  
Alessio Albanese ◽  
Vanessa Modesti ◽  
Alessandro Grottoli ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Material ◽  
Xylella Fastidiosa ◽  
Infected Plant ◽  
Diagnostic Tools ◽  
Nanopore Sequencing ◽  
Direct Dna Sequencing ◽  
Detection And Identification ◽  
Infected Plant Material

SummaryXylella fastidiosa (Xf) is a polyphagous gram-negative bacterial plant pathogen that can infect more than 300 plant species. It is endemic in America while, in 2013, Xf subsp. pauca was for the first time reported in Europe on olive tree in the Southern Italy. The availability of fast and reliable diagnostic tools is indispensable for managing current and future outbreaks of Xf.In this work, we used the Oxford Nanopore Technologies (ONT) device MinION platform for detecting and identifying Xf at species, subspecies and Sequence Type (ST) level straight from infected plant material. The study showed the possibility to detect Xf by direct DNA sequencing and identify the subspecies in highly infected samples. In order to improve sensitivity, Nanopore amplicon sequencing was assessed. Using primers within the set of the seven MLST officially adopted for identifying Xf at type strain level, we developed a workflow consisting in a multiple PCR and an ad hoc pipeline to generate MLST consensus after Nanopore-sequencing of the amplicons. The here-developed combined approach achieved a sensitivity higher than real-time PCR allowing within few hours, the detection and identification of Xf at ST level in infected plant material, also at low level of contamination.Originality Significance StatementIn this work we developed a methodology that allows the detection and identification of Xylella fastidiosa in plant using the Nanopore technology portable device MinION. The approach that we develop resulted more sensitive than methods currently used for detecting X. fastidiosa, like real-time PCR. This approach can be extensively used for X. fastidiosa detection and it may pave the road for the detection of other tedious vascular pathogens.

scholarly journals Detection of Peach latent mosaic viroid by PCR

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.

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

2011 ◽  
Vol 61 (3) ◽  
pp. 489-497 ◽  
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

scholarly journals An Indigenous Virulent Strain of Erwinia amylovora Lacking the Ubiquitous Plasmid pEA29

2006 ◽  
Vol 96 (8) ◽  
pp. 900-907 ◽  
Author(s):  
Pablo Llop ◽  
Victoria Donat ◽  
Margarita Rodríguez ◽  
Jordi Cabrefiga ◽  
Lídia Ruz ◽  
...  
Keyword(s):  
Plant Material ◽  
Fire Blight ◽  
Erwinia Amylovora ◽  
Virulent Strain ◽  
Infected Plant ◽  
Pear Fruit ◽  
Chain Reaction ◽  
Biochemical Tests ◽  
Polymerase Chain ◽  
Infected Plant Material

An atypical strain of Erwinia amylovora was isolated near an outbreak of fire blight at a nursery in Spain in 1996. It was obtained from a Crataegus plant showing typical symptoms and was identified as E. amy-lovora by biochemical tests and enrichment-enzyme-linked immuno-sorbent assay, but not by polymerase chain reaction using primers based on the pEA29 sequence. Nevertheless, with primers from chromosomal regions, the isolate gave the expected amplification band. This strain carries one plasmid of ≈70 kb, with no homology with the 29-kb plasmid common to all pathogenic strains, or with a large plasmid present in some E. amylovora strains. Growth of the strain in minimal medium without thiamine was slower compared with cultures in the same medium with thiamine, a characteristic typical of strains cured of the 29-kb plasmid. Nevertheless, aggressiveness assays on pear, apple, and Pyracantha plants and in immature pear fruit showed that this strain exhibited a virulence level similar to other strains containing pEA29. To the best of our knowledge, this is the first report of the isolation from naturally infected plant material of a pathogenic strain of E. amylovora without pEA29, but with a plasmid of ≈70 kb not previously described.

scholarly journals Potential for dissemination of Phytophthora cinnamomi by feral pigs via ingestion of infected plant material

2013 ◽  
Vol 16 (4) ◽  
pp. 765-774 ◽  
Author(s):  
Andrew Yufa Li ◽  
Nari Williams ◽  
Stanley G. Fenwick ◽  
Giles E. St. J. Hardy ◽  
Peter J. Adams
Keyword(s):  
Plant Material ◽  
Phytophthora Cinnamomi ◽  
Infected Plant ◽  
Feral Pigs ◽  
Infected Plant Material

scholarly journals Modelling the impact and control of an infectious disease in a plant nursery with infected plant material inputs

2016 ◽  
Vol 334 ◽  
pp. 27-43 ◽  
Author(s):  
Andrew M. Bate ◽  
Glyn Jones ◽  
Adam Kleczkowski ◽  
Alan MacLeod ◽  
Rebecca Naylor ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Plant Material ◽  
Infected Plant ◽  
Plant Nursery ◽  
And Control ◽  
The Impact ◽  
Infected Plant Material

scholarly journals Comparative Elimination of Begomoviruses in Cassava Meristems and Axillary Buds

2021 ◽  
pp. 1-9
Author(s):  
Adonise F. Valam Zango ◽  
Innocent Zinga ◽  
Régis Dimitri Longué Soupké ◽  
Simplice Prosper Yandia ◽  
Brice Toko Marabana ◽  
...  
Keyword(s):  
Plant Material ◽  
Central African Republic ◽  
Culture Media ◽  
Effective Means ◽  
Infected Plant ◽  
High Severity ◽  
Rate Of Emergence ◽  
The University ◽  
Infected Plant Material

Aim: The production of healthy cuttings from a local cassava cultivar for cassava mosaic control. Study design: The study was carried out at the In Vitro Culture Laboratory using the techniques of thermotherapy and culture of tissues and explants in a specific medium. Place and duration: The study was carried out at the Laboratory of Biological and Agronomic Sciences for Development at the University of Bangui, Central African Republic from December 2017 to June 2018. Methodology: A variety of cassava called six-month very susceptible to mosaic was used for this work. The cuttings used were infected by Cassava Mosaic Begomoviruses (CMBs) with high severity. It were subcultured in a room under the heat of 37 ° C to 40 ° C for two weeks. Explants and meristems were taken from the stems and the apices, respectively. These collected materials were treated and seeded on appropriate culture media. After the plants produced in vitro were acclimatized and the leaves were removed to check their phytosanitary state by the PCR technique. Results: The rate of emergence of the acclimatized plants and the expression of the disease on the microplants were evaluated. The results show that 75% of the weaned vitro plants recovered under acclimatization. In addition, the acclimatized plants left growing in the greenhouse for four months remained asymptomatic. Molecular analysis by PCR showed that begomoviruses were not detected on meristem samples unlike samples from stem fragments. Conclusion: The combination of thermotherapy technique associated with the culture of meristems constitutes an effective means useful for the sanitation of infected plant material.

Cylindrocarpon musae. [Descriptions of Fungi and Bacteria].

1987 ◽  
Author(s):  
D. Brayford
Keyword(s):  
Costa Rica ◽  
North America ◽  
South America ◽  
Geographical Distribution ◽  
Contaminated Soil ◽  
Plant Material ◽  
Infected Plant ◽  
Long Distance ◽  
Soil P ◽  
Infected Plant Material

Abstract A description is provided for Cylindrocarpon musae. Information is included on the disease caused by the organism, its transmission, geographical distribution, and hosts. HOSTS: Musa AAA (Cavendish). DISEASE: Rotting of fleshy roots and rhizomes of banana. GEOGRAPHICAL DISTRIBUTION: Asia: Philippines; North America: Costa Rica, Guadeloupe, Martinique, Panama; South America: Colombia, Ecuador. TRANSMISSION: The fungus probably survives as 'chlamydospores' in soil. Its slimy spores may be dispersed by water. Long distance spread may potentially occur by transportation of infected plant material or contaminated soil.

scholarly journals First Report of Citrus tristeza virus Infecting Citrus Trees in Georgia, USA

Plant Disease ◽  
2021 ◽  
Author(s):  
Emran Ali ◽  
Allina Bennett ◽  
Tammy Stackhouse ◽  
Sumyya Waliullah ◽  
Jonathan E Oliver
Keyword(s):  
Plant Material ◽  
Citrus Tristeza Virus ◽  
Infected Plant ◽  
Poncirus Trifoliata ◽  
First Report ◽  
Leaf Material ◽  
Tristeza Virus ◽  
Infected Plant Material ◽  
Citrus Tristeza ◽  
Citrus Trees

Citrus tristeza virus (CTV) [genus Closterovirus; family Closteroviridae] is one of the most important, economically devastating viruses of citrus worldwide. On citrus trees grafted onto sour orange rootstock, typical CTV symptoms include dieback and defoliation, stunting, curling and chlorotic leaves, stem-pitting, and pinholes below the bud union on the inner face of the bark (Moreno et al. 2008). This single-stranded, positive-sense RNA virus is most efficiently transmitted by the brown citrus aphid (Toxoptera citricida), but it can also be transmitted by other aphid species and through grafting of infected plant material onto healthy plants (Moreno et al 2008; Herron et al. 2006). In Fall 2020, leaf material for virus testing was collected from 13 navel orange trees (Citrus × sinensis) grafted onto Poncirus trifoliata rootstocks (including ‘Flying Dragon’) located in a citrus research orchard in Tifton, GA. Trees ranged in age from 2 to 10 years, with the younger trees having been grafted from cuttings taken from the older trees. The oldest of these trees was derived from cuttings taken in 2009 from an orange tree growing locally in a residential yard in Tifton; this parent tree was more than 15 years old when these cuttings were obtained and was no longer available for sampling as of 2020. Symptoms or other visible signs of disease had not been noted on any of the tested trees, and trees were chosen for testing prior to the further dissemination of this plant material. The presence of CTV was verified via molecular and serological testing. CTV infection was initially confirmed in 8 of 13 tested samples using the ImmunoStrip® for CTV assay (Agdia® Inc., Elkhart, IN, cat no: ISK 78900/0025) according to the manufacturer’s instructions. RNA was extracted from leaf material collected from the 13 sampled trees using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Following cDNA synthesis, samples were tested for the presence of CTV by reverse-transcription PCR using primer pair AR18F (5’-ATGTCAGGCAGCTTGGGAAATT-3’) and AR18R (5’-TTCGTGTCTAAGTCRCGCTAAACA-3’) which produces a 511 bp amplicon (Roy et al., 2005). PCR reactions confirmed the presence of CTV, with the same eight samples that had previously tested positive via Immunostrip® producing PCR fragments of the expected size. Amplified products from two of these samples were then sequenced using Sanger sequencing (Retrogen Inc, San Diego, CA, USA) and subjected to BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for further identification. Sequence analysis revealed that the obtained partial sequences (MW540805) from the p18 gene of both isolates were 100% identical to one another and shared 100% identity to corresponding sequences from CTV strain N4 (MK779711.1). To the best of our knowledge, this is the first report of CTV infecting citrus plants in Georgia. CTV could pose an imminent threat to the emerging citrus industry in Georgia if it were to become established in commercial citrus plantings either via the dissemination of infected plant material or via vector transfer of the virus under field conditions. While the brown citrus aphid is not known to be widespread in Georgia at this time, other CTV vectors are prevalent including the cotton aphid (Aphis gossypii) and the black citrus aphid (T. aurantia). Georgia citrus growers and plant propagators should be aware of this virus and take appropriate control measures to prevent the spread of this viral diseas.

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

2016 ◽  
Vol 106 (9) ◽  
pp. 1006-1014 ◽  
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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