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Outbreak of leafspot on blackberry ( Rubus fruticosus ) caused by Gnomoniopsis sp . in Iran

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New Disease Reports
Authors:
Hossein Ali Mirhosseini at Gorgan University of Agricultural Sciences and Natural Resources
Hossein Ali Mirhosseini
Heshmat Rahimian at Sari Agricultural Sciences and Natural Resources University
Heshmat Rahimian
Valiollah Babaeizad at Sari Agricultural Sciences and Natural Resources University
Valiollah Babaeizad
Leila Hashemi at Sari Agricultural Sciences and Natural Resources University
Leila Hashemi
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In October 2012, noticeable leaf spots were found on blackberry plants ( Rubus fruticosus ) cultivated in several cities of Mazandaran, a province in northern Iran. Typical lesions observed were light brown, regularly globose and variable in…
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New Disease Reports (2015) 31, 9. http://dx.doi.org/10.5197/j.2044-0588.2015.031.009
Outbreak of leafspot on blackberry (Rubus fruticosus)
caused by Gnomoniopsis sp. in Iran
H.A. Mirhosseini*, H. Rahimian, V. Babaeizad and L. Hashemi
Department of Plant Protection, Sari Agricultural Sciences and Natural Resources University, P.O.BOX 578, Mazandaran,
Sari, Iran
*E-mail: alimirhosseini@yahoo.com
Received: 06 Jan 2015. Published: 08 Mar 2015. Keywords: β-tubulin, Gnomoniopsis idaeicola, ITS-rDNA
Figure 1 Figure 2 Figure 3
Figure 4
In October 2012, noticeable leaf spots were found on blackberry plants
(Rubus fruticosus) cultivated in several cities of Mazandaran, a province in
northern Iran. Typical lesions observed were light brown, regularly globose
and variable in size (Fig. 1). Over time, the lesions enlarged and coalesced,
extending until the entire leaf withered (Fig. 2). The incidence of diseased
plants was approximately 80% in Sari with 30% of the plants extremely
blighted, and yield losses ranging from 50 to 80%. Ten leaves with
symptoms were collected from different blackberry plants from several
fields in Mazandaran. Infected leaf tissues taken from the margins of the
leaf spot were cut into small pieces (5 x 3 mm) and plated onto potato
dextrose agar (PDA) after surface sterilisation in 1.5% NaOCl for three
minutes. The plates were incubated at 24°C for a week. Hyphae growing
out from the tissue pieces were sub-cultured onto PDA. Ten isolates were
obtained and studied for their cultural and morphological characteristics.
Colony colour varied from whitish to grey, olive and dark brown with
moderate to fast growth usually producing abundant conidiomata exuding a
slimy conidial mass (Fig. 3). Conidia were fusiform to slightly obovoid,
straight to slightly curved, one-septate, 5.5-9 x 2-3.5 μm (n=50), which is a
characteristic feature of Gnomoniopsis species (Sogonov et al., 2008).
Total genomic DNA of a representative isolate (GiM68) was extracted
using the CTAB extraction method (Groppe & Boller, 1997) and used for
PCR sequencing of rDNA genes by using universal primers for ITS 1 and
ITS 4 (White et al., 1990) and primers T1/T2 for β-tubulin (TUB2)
(O’Donnell & Cigelnik, 1997). The ITS-rDNA and β-tubulin sequence data
were deposited in GenBank (Accession Nos. KJ563296 and KJ994800,
respectively). A BLAST search in the NCBI GenBank database resulted in
close similarity (98% for ITS-rDNA and 95% for β-tubulin) with G.
idaeicola (KC145891, KC145872, GU320820 for ITS and GU320781 and
GU320782 for β-tubulin). A phylogram was constructed comparing the ITS
and TUB2 sequences of GiM68 with other homologous sequences from the
NCBI GenBank database (Fig. 4). A multiple sequence alignment was
performed using Clustal W and a maximum-likelihood phylogenetic tree
was constructed from Kimura 2-parameter pairwise distances using MEGA
6 (Tamura et al., 2013). Based upon the molecular data and morphological
characteristics, the causal agent was identified as Gnomoniopsis sp., with a
high probability of it being G. idaeicola. Further studies will be conducted
to confirm the species.
A pathogenicity test was conducted on detached, green blackberry leaves.
Fifteen leaves were inoculated by placing a plug (5-mm-diameter) from the
margin of seven-day-old colonies of each of the 10 isolates grown on PDA
on a fresh wound site made with a sterilised scalpel. Each wound was
covered with moisturised cotton and sealed with Parafilm. Control leaves
were inoculated using sterile PDA plugs. The leaves were kept in plastic
bags at ambient temperature (25-30°C). After seven days, all inoculated
leaves with isolates showed symptoms similar to those observed in the field,
whereas the controls remained asymptomatic. The fungus was re-isolated
from the inoculated leaves, fulfilling Koch's postulates. The results of this
study demonstrated Gnomoniopsis sp. as the causal agent of blackberry leaf
spot in Iran for the first time. Previously, G. idaeicola has been reported on
overwintered canes of Rubus spp. in France and USA (Walker, 2012).
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To cite this report: Mirhosseini HA, Rahimian H, Babaeizad V, Hashemi L, 2015. Outbreak of leafspot on blackberry (Rubus fruticosus)
caused by Gnomoniopsis sp. in Iran. New Disease Reports 31, 9. http://dx.doi.org/10.5197/j.2044-0588.2015.031.009
©2015 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 9
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Amplification and direct sequencing of fungal ribosomal genes for phylogenetics protocols. A guide to methods and applications
  • Jan 1990
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White TJ, Bruns T, Lee S, Taylor J, 1990. Amplification and direct sequencing of fungal ribosomal genes for phylogenetics. In: Innis MA, Gelfand DH, Shinsky J, White TJ, eds. PCR protocols. A guide to methods and applications. San Diego, CA, USA: Academic Press, 315-322.
Taxonomy, systematics, ecology, and evolutionary biology of the Gnomoniaceae (Diaporthales), with emphasis on Gnomoniopsis and Ophiognomonia
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