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Peer Reviewed

2011 Plant Management Network.
Accepted for publication 14 April 2011. Published 28 June 2011.

First Report of Stem Dieback and Leaf Spot of Leucothoe caused by Cylindrocladium colhounii in North Carolina

D. M. Benson and K. C. Parker, Department of Plant Pathology, and M. Munster, NCSU Plant Disease and Insect Clinic, Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695; and K. L. Ivors, Department of Plant Pathology, North Carolina State University, Mills River, NC 28759

Corresponding author: D. M. Benson.

Benson, D. M., Parker, K. C., Munster, M., and Ivors, K. L. 2011. First report of stem dieback and leaf spot of Leucothoe caused by Cylindrocladium colhounii in North Carolina. Online. Plant Health Progress doi:10.1094/PHP-2011-0628-01-BR.

Leucothoe fontanesiana (L. catesbaei) and L. axillaris are common in the nursery trade for landscape use in USDA Zones 5 to 8. In the summer of 2009, one-year-old plants growing in 3-gal pots with a poorly-drained potting mix in a North Carolina wholesale nursery exhibited leaf spot and shoot dieback that resulted in over 50% mortality in some blocks of plants (Fig. 1). Isolations on acid PDA from stem and leaf tissue from plants submitted to the clinic resulted in recovery of isolates morphologically identical to Cylindrocladium colhounii Peerally (teleomorph Calonectria colhounii Peerally) (1) (Fig. 2A). This species is not reported on Leucothoe but found on many trees and shrubs worldwide including Pinus strobus in North Carolina, Carya sp. in Virginia, Callistemon rigidus in Florida, Gaultheria procumbens in Oregon, and Ficus carica in Louisiana (2). Morphological characters of the anamorph included 3-septate conidia roughly 58 to 63 m long, and a clavate vesicle on the stipe (Fig 2B). The teleomorph was produced on host tissue after moist-chamber incubation (Fig. 3A). Perithecia were yellow, with the bases turning red when placed in 3% KOH. Ascospores were 3-septate, 49-58 × 5-7 m (Fig. 3B). Isolates also were cultured from symptomatic plants of L. axillaris, L. fontanesiana ‘Girard’s Rainbow,’ and L. fontanesiana ‘Scarletta’ in 3-gal pots collected during a subsequent site visit. Both the internal transcribed spacer region (ITS1, 5.8S, and ITS2) of the ribosomal DNA (rDNA) and part of the β-tubulin gene of two isolates from the original clinic isolation, as well as three isolates collected from plants during the site visit, were sequenced to confirm fungal identification (1,3).


Fig. 1. Symptoms of Cylindrocladium leaf spot and stem dieback caused by C. colhounii on Leucothoe fontanesiana ‘Scarletta’ from the original specimen submitted to the NCSU Plant Disease and Insect Clinic.



Fig. 2. (A) Growth of Cylindrocladium colhounii after re-isolation from an infected plant and transfer to PDA. Left image is through bottom of plate. (B) Conidiogenous apparatus with stipe and vesicle along with conidia of C. colhounii. Scale bar 100 mm.


Fig. 3. (A) Presence of perithecia (globose yellowish spheres) and white mycelium of Calonectria colhounii on an infected stem after incubation in a moist chamber. (B) Ascospores of Calonectria colhounii and single conidium of Cylindrocladium colhounii. Scale bar 50 mm.

Healthy plants with a new flush of growth in 1-gal pots in saucers were inoculated with one of four isolates (an original clinic isolate or one each from the three cultivars) in a greenhouse covered with shadecloth. At inoculation on 25 May 2010, two petri plates of each isolate were blended singly in 250 ml of deionized water, and then screened through several layers of cheesecloth. Fifty milliliters of an individual isolate suspension were sprinkled over the canopy of a test plant with a plastic bottle containing several 3-mm-diameter holes in the lid. Immediately after inoculation, the plants were double bagged: up around the saucer and pot, and then down over the foliage and lower bag. Bags were left in place for 2 days without further irrigation. Two plants of ‘Scarletta’ and one plant each of L. axillaris and ‘Girard’s Rainbow’ were inoculated per isolate, for a total of eight ‘Scarletta’ plants and four plants each of L. axillaris and ‘Girard’s Rainbow’ inoculated. One plant of each cultivar was bagged and left as a non-inoculated control. Plants were arranged in a completely randomized design on the greenhouse bench.

Necrotic leaf and stem symptoms developed over the next 3 weeks with all isolates causing disease. Leaf spots ranged from small individual lesions to larger zonate, necrotic areas that coalesced particularly along the leaf margin. Dieback progressed from stem tips a few centimeters down stems of the youngest growth on some plants (Fig. 4). Disease severity was rated 30 days post-inoculation for each plant on a 1 to 4 scale, where 1 was healthy foliage, 2 was necrosis on a few leaves, 3 was necrosis on many leaves, and 4 was necrosis on most leaves and stem dieback. Disease rating averaged 1.8 for L. axillaris, 2.5 for L. fontanesiana ‘Girard’s Rainbow,’ and 3.0 for L. fontanesiana ‘Scarletta’, respectively, for inoculated plants across the four Cylindrocladium isolates. Symptomatic tissue was collected for re-isolation on acid PDA and C. colhounii was recovered from every inoculated plant but not from the control plants. Sequences of the internal transcribed spacer region of the rDNA and the β-tubulin gene of twelve of the recovered isolates (four from each of the three cultivars) were identical to sequences of the four isolates used for inoculation. Representative sequences of our isolates have been deposited in GenBank (JF742647 and JF742648). This is the first report of C. colhounii attacking L. axillaris and L. fontanesiana in North Carolina.


Fig. 4. Symptoms of Cylindrocladium leaf spot and stem dieback caused by C. colhounii on Leucothoe fontanesiana ‘Scarletta’ 30 days after inoculation: (A) leaf spot and stem dieback in canopy; and (B) close-up of zonate leaf spot and dieback.

Literature Cited

1. Crous, P. W., Groenewald, J. Z., Risde, J-M., Simoneau, P., and Hywel-Jones, N. L. 2004. Calonectria species and their Cylindrocladium anamorphs: Species with sphaeropedunculate vesicles. Stud. Mycol. 50:415-430.

2. Farr, D. F., and Rossman, A. Y. Fungal Databases. Online. Systematic Mycology and Microbiology Laboratory, USDA-ARS, Beltsville, MD.

3. White, T. J., Bruns, T., Lee, S. B., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315-322 in: PCR protocols: A Guide to Methods and Applications. M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White, eds. Academic Press, San Diego, CA.