Blackwell Publishing Ltd
NEW DISEASE REPORT
Plant Pathology (2008) 57, 774
Bacterial leaf spot of tobacco caused by Pseudomonas aeruginosa
in China
L. Yua, X. Y. Qinb, J. Dua, A. Y. Wangb, Y. Y. Zhaoa, D. J. Shena, Y. X. Suna and Q. Huanga*
a
College of Plant Protection, Yunnan Agricultural University, Kunming, 650201, Yunnan; and bThe Department of Plant Protection,
Yunnan Tobacco Science Research Institute, Yuxi, 653100, Yunnan, China
A new disease of tobacco seedlings was observed during 2006 and 2007,
in Xichou and Cangning counties, in Wenshan prefecture and Baoshan city
respectively, in the Yunnan province of China. Symptoms were common
in the float beds and transplants, and first appeared as small, pale or dark
green lesions, sometimes with curving of lower leaves, and brown lesions
on the stems. Sometimes a wet rot developed, or irregularly shaped brown
areas with yellow haloes developed on older leaves, with some plants
eventually dying. These symptoms were consistent with those of Philippine
bacterial leaf spot described by Shew & Lucas (1991).
A fluorescent bacterium was isolated from diseased tissues onto King’s
medium B. Twelve strains were tested according to Schaad (1980). They
produced positive reactions in tests for oxidase, arginine dihydrolase,
nitrate reduction, growth at 41°C, and utilisation of mannitol, geraniol, and
benzoate. Negative reactions were obtained for utilisation of cellobiose,
sorbitol, trehalose, sucrose, m-tartrate, D-tartrate, D-arabinose, L-rhamnose.
On this basis they were identified as Pseudomonas aeruginosa. Meta-
bolic profiling (Biolog Station ELX808BLG, Biolog Microlog™ 4·20 database
and software) indicated 0·51–0·72 similarity to P. aeruginosa.
Molecular identification was done according to Liu et al. (2002). PCR
amplification was done with primers UFPL (5′-AGTTTGATCCTGGCTCAG-
3′) and URPL (5′-GGTTACCTTGTTACGACTT-3′). The sequence of the
1491 bp product (GenBank Acc. No. AM930266) had 98% similarity to
16S rDNA sequences of P. aeruginosa (AY486350) by using BLAST.
Pathogenicity of five strains was confirmed on 4-week-old tobacco seedlings
by either infiltrating leaves or injecting stems with a suspension containing
*E-mail: huangqiong88hs@yahoo.com.cn. Accepted 27 February 2008 at www.bspp.org.uk/ndr where figures relating to this paper can be viewed.
Plant Pathology (2008) 57, 774
Erwinia papayae causing papaya dieback in Malaysia
N. H. Maktara, S. Kamisb, F. Z. Mohd Yusofc and N. H. Hussaina*
a
Institute of Science, Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor; bPlant Protection and
Quarantine Division, Dept. of Agriculture, Jalan Gallagher, 50632, Kuala Lumpur; and cFaculty of Applied Sciences, Universiti Teknologi
MARA, 40450 Shah Alam, Selangor, Malaysia
A papaya die-back disease was first reported in Malaysia by the Johor State
Department of Agriculture in 2003. By the end of 2006 it had spread to
five other states on the west coast of the Malaysian peninsula, affecting
ca. 800 ha and resulting in the destruction of ca. 1 million trees. Total yield
losses were estimated at 200 000 metric tonnes, equivalent to US$58 million.
The varieties affected were Eksotika, Solo, Hong Kong and Sekaki. Early
symptoms included yellowing and necrosis along leaf edges followed by
water-soaked areas on the bases of leaf stalks, crowns and along leaf
mid-ribs. Fruit symptoms included dark spots on the skin and water-soaked
flesh. Later, necrotic and water-soaked areas developed on stems and
spread to the internal tissues, followed by secondary fungal infections. In
advanced stages bending of water-soaked leaf stalks occurs, leading to
dieback, and death of trees.
Hyaline bacterial colonies were consistently isolated on Luria Bertani
agar from infected leaves, crowns, leaf midribs, fruits and leaf stalks after
incubation for 48 h at 30°C. Isolates were Gram-negative rods, facultative
anaerobes, oxidase negative and catalase positive. Six isolates were tested
for tobacco hypersensitivity (Lelliott & Stead, 1987) and gave a strong
response within 10–12 h. Pathogenicity was tested by inoculating suspen-
sions into crowns and leaf stalks of 5-month-old healthy papaya plants (cv.
Eksotika). Sterile distilled water was used as a negative control. Characteristic
symptoms were observed one week after inoculation. No symptoms were
observed on control plants. The original bacterium was re-isolated from
plants showing symptoms and one isolate was sent to a commercial service
for 16S rRNA gene sequencing. The isolate was identified as Erwinia papayae
based on BLAST analyses of sequences in the NCBI database, with the
highest similarity to E. papayae (GenBank Acc No: AY131237·1) from the
*E-mail: noorhana@salam.uitm.edu.my. Accepted 27 February 2008 at www.bspp.org.uk/ndr where figures relating to this paper can be viewed.
774
Doi: 10.1111/j.1365-3059.2008.01876.x
approx. 6 × 106 CFU mL–1. Sterile water was used as a control. A wet rot,
curved leaves, and severe necrotic symptoms developed on inoculated
plants within 7–21 days. No symptoms developed on control plants. The
bacterium was re-isolated from inoculated plants. The bacterium was con-
firmed as P. aeruginosa on the basis of the above characteristics. Bacterial
leaf spot caused by P. aeruginosa has been reported previously in the
Philippines (Shew & Lucas, 1991), but this is the first report of the disease
from China.
Acknowledgements
This work was supported by Yunnan provincial tobacco company, China
(No: 2006A10).
References
Liu L, Coenye T, Burns JL, Whitby PW, Stull TL, LiPuma JJ, 2002. Ribosomal
DNA-directed PCR for identification of Achromobacter (Alcaligenes)
xylosoxidans recovered from sputum samples from cystic fibrosis patients.
Journal of Clinical Microbiology 40, 1210–3.
Schaad NW, 1980. Pseudomonas. In: Schaad NW, ed. Laboratory Guide for
Identification of Plant Pathogenic Bacteria. St. Paul, MN, USA: APS Press,
36–44.
Shew HD, Lucas GB, 1991. Philippine bacterial leaf spot. In: Lucas GB, ed.
Compendium of Tobacco Diseases. St. Paul, MN, USA: APS Press, 33.
Doi: 10.1111/j.1365-3059.2008.01877.x
Caribbean (Gardan et al., 2004). PCR using phytoplasma specific primers,
P1and P7 (Guthrie et al., 1998) ruled-out the involvement of phytoplasma.
This is the first report of E. papayae causing papaya dieback in Malaysia.
The disease may have entered Malaysia via Johor, then spread to other
states by human activity and possibly birds and insects. The first report of
this disease in S.E. Asia was in Java (von Rant, 1931). Erwinia papayae
was first reported as causing bacterial canker of papaya in the Caribbean
by Gardan et al. (2004).
Acknowledgements
The authors would like to thank the Plant Protection and Quarantine
Division, Department of Agriculture, Malaysia and the Faculty of Applied
Sciences, UiTM.
References
Gardan L, Christen R, Achouak W, Prior P, 2004. Erwinia papayae sp. nov., a
pathogen of papaya (Carica papaya). International Journal of Systematic and
Evolutionary Microbiology 54, 107–13.
Guthrie JN, White PT, Walsh KB, Scott PT, 1998. Epidemiology of
phytoplasma associated papaya diseases in Queensland, Australia. Plant
Disease 82, 1107–11.
Lelliott RA, Stead DE, 1987. Methods for the Diagnosis of Bacterial Diseases
of Plants. Oxford, UK: Blackwell Scientific Publications.
von Rant, A, 1931. Über eine Bakterienkrankheit bei dem Melonenbaume
(Carica papaya L.) auf Java. Zentralblatt für Bakteriologie Parasitenkunde
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© 2008 The Authors
Journal compilation © 2008 BSPP