300

Journal of Plant Pathology (2017),

99 (1), 287-304

Received January 8, 2017
Accepted February 14, 2017

Corresponding author: A. Perek
E-mail: [e-mail]

Received December 16, 2016
Accepted January 11, 2017

Corresponding author: S. Iftikhar
E-mail: [e-mail]

D

isease

N

ote

FIRST REPORT ON

MYCOSPHAERELLA

MORI

ON

MORUS NIGRA

AND

M. RUBRA

IN POLAND

K. Pieczul, E. Jajor, A. Perek and I.

Ś

wierczy

ń

ska

Department of Mycology, Institute of Plant Protection – National

Research Institute, ul. W

ę

gorka 20, 60-318 Pozna

ń

, Poland

In 2016 leaf spot and necrosis were observed on mulberry

plants (Morus nigra and M. rubra) in house gardens and pub-

lic parks in the city of Pozna

ń

and in several other locations

in the west of Greater Poland district. At first, evenly dis-

tributed small, brown spots appeared on the leaves, which

successively developed in oval or irregular necrotic lesions

up to 18 mm in size. Infected tissues from M. nigra, M. ru-
bra and M. alba collected from eight locations were cut to

small pieces, surface-sterilized and transferred to Potato

Dextrose Agar (PDA). Cultures incubated at 21°C for 10

days exhibited a dense, velvety, whitish to grey mycelium.

Hyaline, straight or curved conidia, mostly 3-6 septate 54

(38-82) × 4.4 (4-5) μm were observed either on affected leaves

or on isolates grown on PCA (Potato carrot agar). Pathoge-

nicity tests were conducted according to Hong et al. (2011)

on three M. alba plants with two isolates from M. nigra and
M. rubra. Small brown spots developed only on inoculated

leaves, 12 dpi, from which the same fungus used for inocula-

tions was reisolated. The internal transcribed spacer region

of 10 cultures (five M. alba, three M. nigra and two M. rubra)

was PCR-amplified followed by sequencing of the amplified

products (GenBank accession Nos. KX982229-31). A BLAST

search revealed 100% identity of all tested amplicons with

the sequence of Mycosphaerella mori (AB435069). Molecular

analysis and morphological features, support the identifica-

tion of M. mori as the causal agent of the disease observed

on M. nigra and M. rubra. This pathogen is of wide occur-

rence on Morus spp. in the world (Farr and Rossman, 2017).

To our knowledge this is the first report of M. mori on M.
nigra and M. rubra in Poland.

Farr D.F., Rossman A.Y., 2017. Fungal Databases, Systematic My-

cology and Microbiology Laboratory, ARS, USDA. Available

from: http://nt.ers-grin.gov/fungaldatabases.

Hong S.K., Kim W.G., Sung G.B., Choi H.W., Lee Y.K., Shim H.S.,

Lee S.Y., 2011. Occurrence of leaf spot on mulberry caused by

Phloeospora maculans in Korea. Plant Pathology Journal

27: 193.

D

isease

N

ote

FIRST REPORT OF

ALTERNARIA

ALTERNATA

CAUSING BROWN LEAF

SPOT OF POTATO IN PAKISTAN

A.A. Shahid

1,2

, S. Iftikhar

1

, K. Nawaz

1

,

W. Anwar

1

and M. Ali

1

1

Institute of Agricultural Sciences,

University of the Punjab, Lahore, Pakistan

2

Center of Excellence in Molecular Biology,

University of the Punjab, Lahore, Pakistan

Potato plants with brown spot symptoms were observed in

the Punjab, Pakistan during March 2015, with an approximat-

ed incidence of 45.5%. Symptoms were small, brown lesions

on leaves with concentric rings coalescing into larger lesions.

Infected leaves died. For pathogen isolation, surface sterilized

leaves were cut from lesion edges, and incubated at 25 ± 2°C

on potato dextrose agar medium for 7 days. Fungal colonies

were fast-growing, brownish, and cottony. Conidiophores

arised singly or in clusters, usually 2-6, 42-27 μm in length

and 4-25 μm in width. Conidia were greenish brown, catenate,

ovoid or obclavate, multi-celled, with 2-6 transverse septa, 1-2

longitudinal septa, 12-32 × 6-12 μm in size. Genomic DNA of

the fungus was isolated by CTAB method, amplified using

ITS1 and ITS4 primers (White et al., 1990), sequenced (Gen-

Bank Accession No. LT605000) and showed 99% similarity

to the sequences of Alternaria alternata isolates. Pathogen was

identified as Alternaria alternata (Fr.) Keissler. Koch’s postu-

lates were verified with modified in vitro detached leaves assay

(Park et al., 2008). Fifteen potato leaflets were placed with

the bottom side up in a Petri dish, then covered with paper

towels and 20 ml distilled water. Spore suspension (1 × 10

5

) of

A. alternata from 7 day-old cultures was drop inoculated on

each leaf side; sterile water was used for control leaves. The

experiment was repeated twice. Inoculated leaves stored at

25 ± 2°C for 7 days, developed brown lesions and spots similar

to those observed in the field, while control leaves showed

no symptoms. A. alternata was re-isolated from inoculated

leaves and pathogenicity test confirmed it as the casual agent

of brown spot disease. Brown leaf spot caused by A. alternata

on different hosts was also reported in South Africa and other

parts of the world (Van der Waals et al., 2011; Thomma, 2003).

This is the first report of A. alternata causing brown spot of

potato in Pakistan.

Park D.S., Sayler R. J., Hong Y.G., Nam M.H., Yang Y., 2008. A

method for inoculation and evaluation of rice sheath blight

disease. Plant Disease

92: 25-29.

Thomma B.P.H.J., 2003. Alternaria spp.: from general saprophyte

to specific parasite. Molecular Plant Pathology

4: 225-236.

Van der Waals J. E., Pitsi B. E., Marais C., Wairuri C.K., 2011.

First report of Alternaria alternata causing brown leaf spot of

potatoes in South Africa. Plant Disease

95: 363-366.

White T.J., Bruns T., Lee S., 1990. Amplification and direct se-

quencing of fungal ribosomal RNA genes for phylogenetics.

In: Taylor J., Innis A., Gelfand D.H., Sninsky J.J. (eds). PCR

Protocols, pp. 315-322. Academic Press, San Diego, CA, USA.