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First Report of Stemphylium vesicarium as Causal Agent of Wilting and Root Rotting of Radish Sprouts in Italy

Authors:
Alessandra Belisario at Council for Agricultural Research and Agricultural Economy Analysis
Alessandra Belisario
Salvatore Vitale at Council for Agricultural Research and Agricultural Economy Analysis
Salvatore Vitale
Laura Luongo at Council for Agricultural Research and Agricultural Economy Analysis
Laura Luongo
S. Nardi
S. Nardi
S. Nardi
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Abstract and Figures

A consistent contamination from a Stemphylium sp. was detected on radish (Raphanus sativus) seeds by a seed blotter test. Twenty-five percent of seed lots were contaminated. Stemphylium vesicarium (teleomorph Pleospora allii) was identified on the basis of morphological characters of conidia and conidiophores (4). Conidia were golden brown to dark drown, oblong to oval with one to four transverse and one to three longitudinal septa, constricted at one to three of the major transverse septa. Conidia dimensions ranged from 12 to 22 × 30 to 40 μm. Conidiophores were straight or occasionally one-branched with a swollen apex and one to four septate. Pseudothecia with asci and ascopores were observed on radish seeds. Asci were cylindrical to clavate with eight ascospores with up to six transverse septa and numerous longitudinal septa. Species identification was also confirmed after comparing the sequences of the internal transcribed spacer (ITS) region of rDNA and gpd (glyceraldehyde-3-phosphate dehydrogenase) (3) of four isolates with those of Stemphylium species already present in the NCBI database. Accessions Nos. AM 746020 to AM746023 and AM883174 to AM883177 were deposited for ITS and gpd, respectively. Artificial inoculations were carried out on radish seeds previously disinfected with 1% sodium hypochlorite for 10 min and then plated on S. vesicarium sporulating colonies grown on potato dextrose agar (PDA). The four sequenced isolates were tested for pathogenicity. Disinfected seeds were plated onto PDA only and used as a control. After 48 h of incubation, seeds were sown in sterilized soil in plastic plates. The emerging and the eventually dead plants were counted. Stem necrosis and root rotting developed on sprouts within the first week after sowing. On the surviving infected plantlets, wilting and death occurred on more than 70% of the plants within 4 weeks after sowing. Control plantlets obtained from disinfected seeds remained healthy. The fungus reisolated from wilted and dead plants was morphologically identical to the original isolates, thus confirming S. vesicarium as the causal agent. In Italy, this pathogen is common on asparagus (1), but it has also been reported on Allium spp., tomato, and pear. On European pear it is the causal agent of brown spot (2), a destructive disease in the Mediterranean area but also in the Netherlands and other continental European countries. On the basis of these results, seed contamination with S. vesicarium can represent a threat for the production of radish for sprout consumption. To our knowledge, this is the first report of S. vesicarium on radish plantlets in Italy. References: (1) F. Del Zan et al. L'informatore Agrario 11:95, 1989. (2) I. Llorente and E. Montesinos. Plant Dis. 90:1368, 2006. (3) B. M. Pryor and D. M. Bigelow. Mycologia 95:1141, 2003. (4) E. G. Simmons. Sydowia 38:284, 1985.
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New Disease Reports (2017) 35, 36. http://dx.doi.org/10.5197/j.2044-0588.2017.035.036
First report of Stemphylium vesicarium on chilli pepper
in Italy
S. Vitale, L. Luongo, M. Galli and A. Belisario*
Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria, Centro di Ricerca Difesa e Certificazione (CREA-
DC), Via C.G. Bertero, 22 - 00156 Rome, Italy
*E-mail: alessandra.belisario@crea.gov.it
Received: 07 Jun 2017. Published: 21 Jun 2017. Keywords: Capsicum spp., glyceraldehyde 3-phosphate, grey leaf spot, ITS
Figure 1 Figure 2 Figure 3
Figure 4
In 2014 during a survey of fields of chilli pepper, grey leaf spot (GLS)
symptoms were observed on leaves in two different Italian areas, Rieti
(central Italy) and Cosenza (southern Italy). Diseased leaves were
characterised by numerous tiny round spots (0.5 to 2.5 mm diameter,
average 1.3 mm) that were white to grey with a sunken centre and brown
edge, resulting in premature defoliation (Fig. 1). No other part of the plant
was affected. The varieties 'Fatalii White' (Capsicum chinense) and
'Diavolicchio Calabrese' (C. annum) were the most susceptible in central
and southern Italy, respectively. In both locations disease incidence on the
two chilli pepper varieties was >40%.
Tissue from leaves with typical GLS symptoms was plated on potato
dextrose agar amended with streptomycin and ampicillin (100 ppm each)
and incubated at 22 ±0.5°C with a 12 hour photoperiod. Single spore
isolations were performed to obtain pure cultures (Fig. 2) and the
morphological characteristics were consistent with the type description of
Stemphylium vesicarium (Wallroth) Simmons (Simmons, 1969). To confirm
morphological identification, a representative isolate from each of the two
Italian areas (ISPaVe2162 and ISPaVe2165) were subjected to molecular
analysis. The internal transcribed spacer (ITS) and glyceraldehyde
3-phosphate dehydrogenase (gpd) gene were amplified using universal
primers ITS5 and ITS4 (White et al., 1990) and gpd1 and gpd2 (Berbee et
al., 1999), respectively and sequences were deposited in the European
Nucleotide Archive (Accession Nos. LN896692 and LN896693 for
the ITS, and LN896694 and LN896695 for the gpd gene, respectively). A
BLAST search in GenBank showed 100% identity with S. vesicarium in
both the ITS region (JX424810) and gpd gene (DQ000654). To further
support identification, phylogenetic analyses using the Maximum
Composite Likelihood method (Kimura2/parameter model) was performed
(MEGA 5.2). The isolates ISPaVe2162 and ISPaVe2165 clustered together
with S. vesicarium isolates (Fig. 3).
Pathogenicity tests of the two isolates were performed by artificial
inoculations on detached leaves of the chilli pepper varieties 'Diavolicchio
Calabrese' and 'Fatalii White'. A 10 μl drop of a 1 × 105conidia/ml
suspension was placed on the abaxial leaf surface. A 10 μl drop of sterile
distilled water was used as negative control. The inoculated leaves were
incubated in sterile Petri plates, 20 mm in diameter, containing water-
saturated sterile blotting paper at room temperature (24 ±2°C). Brown
lesions started to be visible on detached leaves three days after inoculation.
Subsequently, lesions enlarged in size and typical GLS were evident at all
inoculation points on both chilli pepper varieties (Fig. 4). No symptoms
developed on the leaf inoculated with sterile distilled water.
In Italy, S. vesicarium has been reported as the causal agent of leaf spots on
tomato (Porta-Puglia et al., 2001) and wilting and root rot on radish sprouts
(Belisario et al., 2008). To our knowledge this is the first report of S.
vesicarium on chilli pepper in Italy. The presence of S. vesicarium in two
distinct geographic areas in Italy suggests an association with seed
contamination and Stemphylium spp. are generally seed-borne.
Consequently more strict control of seed health for chilli pepper production
would be advisable.
References
Belisario A, Vitale S, Luongo L, Nardi S, Talevi S, Corvi F, 2008. First
report of Stemphylium vesicarium as causal agent of wilting and root rotting
of radish sprouts in Italy. Plant Disease 92, 651.
http://dx.doi.org/10.1094/PDIS-92-4-0651C
Berbee ML, Pirseyedi M, Hubbard S, 1999. Cochliobolus phylogenetics and
the origin of known, highly virulent pathogens, inferred from ITS and
glyceraldehyde-3-phosphate dehydrogenase gene sequences. Mycologia 91,
964-977. http://dx.doi.org/10.2307/3761627
Porta-Puglia A, Conca G, Ortu P, 2001. Stemphylium vesicarium, patogeno
poco noto del pomodoro. L'Informatore Agrario 40, 73-74.
Simmons EG, 1969. Perfect states of Stemphylium. Mycologia 61, 1-26.
http://dx.doi.org/10.2307/3757341
White TJ, Bruns T, Lee S, Taylor JW, 1990. Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis
MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR Protocols: A Guide to
Methods and Applications, New York, USA: Academic Press, 315-322.
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To cite this report: Vitale S, Luongo L, Galli M, Belisario A, 2017. First report of Stemphylium vesicarium on chilli pepper in Italy. New
Disease Reports 35, 36. http://dx.doi.org/10.5197/j.2044-0588.2017.035.036
©2017 The Authors This report was published on-line at www.ndrs.org.uk where high quality versions of the figures can be found.
New Disease Reports is a peer-reviewed on-line journal published by the British Society for Plant Pathology,
for more information visit http://www.ndrs.org.uk/ Page 36
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Article
  • Nov 1999
We evaluate the phylogenetic distribution of known, highly virulent plant pathogens in the genus Cochliobolus (sexually reproducing species in the Ascomycota, Pleosporaceae) and assess the relationship between Cochliobolus species and species of Curvularia and Bipolaris (asexual states of fungi in the Ascomycota, Pleosporaceae). To infer a phylogeny, we have used two sequence regions: (i) the complete ITS 1, ITS 2, and 5.88 rDNA sequences for 65 fungal isolates and (ii) a 600 bp fragment of the housekeeping gene gpd, coding for glyceraldehyde-3-phosphate dehydrogenase, for 54 isolates. We combined ITS, 5.8S and gpd sequence data from 41 species. Ln the Cochliobolus clade, 31 out of 32 species fit clearly into one of two groups. One species, Cochliobolus homomorphus, did not fit clearly into either group. The 13 species in Cochliobolus Group I grouped together with 100% bootstrap support from the combined ITS/gpd data. This group included Cochliobolus and Bipolaris species that cause serious crop losses, such as Co. sativus, Co. miyabeanus, Co. carbonum and Co. heterostrophus. However; within Group 1, the known, highly virulent pathogens did not form a monophyletic group of species. Average substitution levels between pairs of species in the Group 1 were low, about 1.7% in the ITS region, suggesting that these species had radiated rapidly and recently. The 18 species in Cochliobolus Group 2 formed a monophyletic group in 96% of parsimony bootstrap replicates of the combined ITS and gpd data. The Cochliobolus species that were transferred into the segregate genus Pseudocochliobolus were in this second group. This study included 9 Curvularia and Bipolaris species without known sexual states and they all appear to be recently derived from among sexual species of Cochliobolus. Both Curvularia and Bipolaris were polyphyletic, but only Bipolaris states were associated with Group 1 Cochliobolus species. Both Curvularia and Bipolaris states were associated with species in Cochliobolus Group 2.
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Stemphylium vesicarium, patogeno poco noto del pomodoro
  • Jan 2001
  • 73-74
  • A Porta-Puglia
  • G Conca
  • P Ortu
Porta-Puglia A, Conca G, Ortu P, 2001. Stemphylium vesicarium, patogeno poco noto del pomodoro. L'Informatore Agrario 40, 73-74.