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© 2013 Plant Management Network.
Accepted for publication 18 May 2013. Published 29 July 2013.


First Report of Pantoea ananatis (Syn. Erwinia uredovora) Being Associated with Peanut Rust in Georgia


R. S. Arias, USDA-ARS National Peanut Research Laboratory, 1011 Forrester Drive SE, Dawson, GA 39842; I. L. Power and A. K. Culbreath, University of Georgia, Tifton Campus, 2360 Rainwater Road, Tifton, GA 31793; and V. S. Sobolev and M. C. Lamb, USDA-ARS National Peanut Research Laboratory, 1011 Forrester Drive SE, Dawson, GA 39842


Corresponding author: R. S. Arias.  renee.arias@ars.usda.gov


Arias, R. S., Power, I. L., Culbreath, A. K., Sobolev, V. S., and Lamb, M. C. 2013. First report of Pantoea ananatis (syn. Erwinia uredovora) being associated with peanut rust in Georgia. Online. Plant Health Progress doi:10.1094/PHP-2013-0729-04-BR.


Peanut rust (Figs. 1 and 2) and leaf spots are the main foliar fungal diseases of peanut (Arachis hypogaea L). Peanut rust occurs every year in Texas, and sporadically in Alabama (6) and Georgia. Fungicide applications to control peanut leaf spots caused by Cercospora arachidicola Hori and Cercosporidium personatum (Berk & M.A. Curtis) Deighton are also effective against peanut rust. However, untreated, peanut rust could be as devastating as leaf spot diseases (6) causing severe defoliation. To genetically characterize the causal agent of peanut rust, Puccinia arachidis Speg., we prepared SSR-enriched libraries according to the method of Techen et al. (7) adapted for Nextera kit (Epicentre, Madison, WI) using DNA of urediniospores, and performed high-throughput sequencing with Roche 454 as previously described (2). Surprisingly, after assembling contigs and comparing sequences to databases using BLAST analysis (1), almost half of the contigs had significant similarity (expected values ≤ 1 × 10-4) to the bacterium Pantoea ananatis (Syn. Erwinia uredovora). To confirm the presence of this bacterium, we used urediniospores collected from three locations in Georgia, one from Attapulgus and two from Tifton (Fig. 3). Ten-fold dilutions of P. arachidis urediniospores were made in phosphate-buffer saline (PBS), determined urediniospore number in the suspensions using a haemocytometer, and plated the dilutions onto Luria-Bertani (3) Agar plates. Bacterial colonies growing on plates from the Attapulgus sample had the appearance of a pure culture, with yellow-rounded glossy colonies like Erwinia (Fig. 4); these colonies were counted and estimated at 30 cfu per urediniospore. The same type of colonies were observed in the Tifton samples, but at much lower proportion compared to other bacterial species on the Petri dishes. To confirm the identity of the Erwinia-like bacteria growing on Petri dishes, a total of 52 colonies were isolated, including some not similar to Erwinia spp., and a 1300-bp fragment of their 16S ribosomal-RNA genes was PCR amplified. PCR amplicons were cloned into TOPO4 vector (Invitrogen) and sequenced. BLAST analysis confirmed that colonies resembling Erwinia were indeed P. ananatis. P. ananatis has received many scientific names throughout the years, i.e., Pectobacterium ananas (Serrano), then, renamed as Erwinia uredovora (Pon) Dye 1963 was reported as parasitic of uredia in cereal rust. The term "uredo-vora" means "feeds on uredospores," and in 1975 Hevesi and Mashaal described the mechanism of infection of E. uredovora as a parasite of fungi. The potential of E. uredovora as biological control of rust-producing fungal pathogens, by inhibiting urediniospore germination and lysing spores of Puccinia graminis, P. recondita, P. coronata, and Uromyces appendiculatus was summarized by Gowdu and Balasubramanian (5). Other agricultural benefits of P. ananatis (Syn. E. uredovora) are: as plant-growth-promoting rhizobacterium (PGPR) in pepper and potato; biological control of insect pests; E. uredovora was the source of the β-carotene pathway that was transferred to rice (Golden rice) to synthesize β-carotene (provitamin-A) and improve its nutritional value (4). On the adverse effects, P. ananatis can cause diseases in crops, such as the fruitlet brown rot in pineapple (Ananas comosus L.) and leaf spot disease in maize (Zea mays L.), and is spreading to new hosts (4). The presence of P. ananatis in peanut rust should be taken in consideration: (i) as it could compromise the viability of urediniospores used for testing resistance of peanut cultivars; (ii) because this bacterium is infecting new plant hosts; and (iii) because of its potential use as biological control of peanut rust.


 

Fig. 1. Symptoms of peanut rust caused by Puccinia arachidids. Note the uredinia on the abaxial surface of the leaflets.

 

Fig. 2. Urediniospores of Puccinia arachidids.


     
 

Fig. 3. Map of Georgia showing the locations from where samples were collected, Attapulgus and Tifton.

 

Fig. 4. Luria-Bertani plate showing bacterial growth on a 10-4 dilution of urediniospores collected from Attapulgus, GA. The highly abundant yellow, smooth, shiny colonies resulted on >3,800 significant hits for the species Pantoea ananatis during BLAST analysis of high throughput sequencing, and were again confirmed as this species by cloning and sequencing of their 16S rRNA gene in the present work.

 


Literature Cited

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2. Arias, R. S., Borrone, J. W., Tondo, C. L., Kuhn, D. N., Irish, B. M., and Schnell, R. J. 2012. Genomics of tropical fruit tree crops. Pages 209-239 in: Genomics of Tree Crops. R. J. Schnell and P. M. Priyadarshan, eds. Springer, New York, NY.

3. Bertani, G. 1952. Studies on Lysogenesis, I: The mode of phage liberation by lysogenic Escherichia coli. J. Bateriol. 62:293-300.

4. Coutinho, T. A., and Venter, S. N. 2009. Pantoea ananatis: An unconventional plant pathogen. Mol. Plant Pathol. 10:325-335.

5. Gowdu, B. J., and Balasubramanian, R. 1988. Role of phylloplane microorganisms in the biological-control of foliar plant diseases. Z. Pflanzenkrankh. (Pflanzenpathol.) Pflanzenschutz. 95:310-331.

6. Hagan, A. 1998. Foliar diseases of peanut. Alabama Cooperative Extension System ANR-369. Alabama A&M University, Normal, AL, and Auburn University, Auburn, AL.

7. Techen, N., Arias, R. S., Glynn, N. C., Pan, Z., Khan, I. A., and Scheffler, B. E. 2010. Optimized construction of microsatellite-enriched libraries. Mol. Ecol. Res. 10:508-515.