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2009 Plant Management Network.
Accepted for publication 11 March 2009. Published 13 April 2009.


Collar Rot on Italian Alder Trees in California Caused by Phytophthora siskiyouensis


Suzanne Rooney-Latham, Associate Plant Pathologist, and Cheryl L. Blomquist, Senior Plant Pathologist, Plant Pest Diagnostics Center, California Department of Food and Agriculture, Sacramento, CA 95832; Tomas Pastalka, Associate Agricultural Biologist, California Department of Food and Agriculture, San Francisco, CA 94107; and Laurence Costello, Environmental Horticulture Advisor, University of California Cooperative Extension San Mateo-San Francisco Counties, Half Moon Bay, CA 94019


Corresponding author: Suzanne Rooney-Latham. slatham@cdfa.ca.gov


Rooney-Latham, S., Blomquist, C. L., Pastalka, T., and Costello, L. 2009. Collar rot on Italian alder trees in California caused by Phytophthora siskiyouensis. Online. Plant Health Progress doi:10.1094/PHP-2009-0413-01-RS.


Abstract

In November 2006, trees of Italian alder (Alnus cordata) were observed declining in association with bleeding trunk cankers in a commercial landscape in Foster City, CA. A species of Phytophthora was isolated on PARP selective medium from the leading edge of the cankers. The Phytophthora species was homothallic with primarily paragynous antheridia and had oospores that were mostly globose and aplerotic. Sporangia were produced from mycelia on plugs of carrot piece agar in soil extraction solution and were semi-papillate and ovoid to ellipsoid in shape. The intergenic transcribed spacer region of rDNA from an alder isolate matched with 100% identity to isolates in GenBank of Phytophthora siskiyouensis, a recently described species associated with tanoak and found in the soil and waterways of coastal Oregon. Pathogenicity was tested on young alder trees growing in pots. Pathogenicity was confirmed on Italian alder trees and potential pathogenicity was demonstrated on red and white alder trees.


Introduction

Phytophthora species cause cankers on the stems of many hardwood trees in forests and landscapes (6). In November 2006, we were contacted about a grove of dying Italian alder trees (Alnus cordata) in San Mateo Co., CA. The trees were approximately 10 years old and located in Foster City (Fig. 1A). Approximately one-half of the 50 trees at the site had died. Some of the dead trees had been removed due to safety concerns, but many were still standing (Fig. 1B). Landscapers reported that the trees had begun to show symptoms 2 to 3 years previously. The predominant symptoms on the remaining trees were sparse foliage, dieback in the canopy, and bleeding cankers on the trunks. Cankers occurred primarily at the bases of the trunks near the soil line and extended upward; a few trees had isolated cankers up to 2 m above the soil surface (Figs. 1C and D). Not all trees exhibiting dieback had cankers. When the outer bark was removed from the cankers, a cinnamon-brown margin was observed separating cream-colored healthy tissue from dark orange-brown diseased tissue. The diseased tissue extended through the bark to the vascular cambium and sapwood interface, characteristic of Phytophthora diseases (Fig. 2) (6). Sprinkler emitters were located around the bases of most of the trees, and many of the emitters were pointed directly at the trunks and bleeding cankers (Fig. 1C, arrow).


 
A
 
B
 
 

C
 

D
 
 

Fig. 1. Phytophthora collar rot on Italian alder trees: (A) grove of dying trees in a commercial landscape in Foster City, CA; (B) standing, dead tree; (C) bleeding canker at the base of a tree and a sprinkler emitter (arrow) adjacent to the trunk; (D) an isolated bleeding canker on the trunk located approximately 2 m above the ground.

 

 
A

 

B
 
C

Fig. 2. Bole lesions in the tissues under the bark of a bleeding canker: (A) distinct margin between healthy and disease tissues; (B) close-up of margin area; (C) discoloration in the secondary phloem tissue.


Data Collection and Pathogenicity Experiments

Samples were collected in November 2006 and January 2007 from seven symptomatic trees. In the field, small pieces of bark and discolored vascular tissue from the margins of the cankers were placed on plates of PARP selective medium (2). Cultures were incubated in the dark at room temperature (18 to 23C) for approximately 7 days. Samples of feeder roots were collected from the affected trees, washed, cut into 1-cm lengths and cultured onto PARP. In January, four soil samples were collected under two trees with extensive symptoms. Soil samples were assayed for Phytophthora spp. in the laboratory by baiting. One soil sample (500 ml) from each tree was placed in a resealable 26.8 by 27.3 cm plastic bag and then flooded with deionized water. Unripe DAnjou pears were washed gently and immersed halfway in the soil-water mixture. Bags were sealed and placed at room temperature for 3 days. Pears were removed from the bags, rinsed and held at room temperature for another 3 days. Tissue pieces from lesions that developed on the pears were placed on PARP medium and plates were placed in the dark at room temperature. Plates were examined for growth for approximately 7 days.

A single colony type consistently grew from trunk pieces taken from four of the seven symptomatic trees. These colonies appeared to be from a single species of Phytophthora. The Phytophthora sp. was isolated only from pieces taken from the vascular cambium and sapwood interface and was not isolated from discolored bark pieces. Isolates were subcultured onto plates of water agar containing organic carrot pieces (CPA) to induce the production of sexual and asexual structures (8). The species of Phytophthora was homothallic, produced aplerotic and globose oospores in approximately 2 weeks, and had mostly paragynous antheridia (Fig. 3A). Oospores averaged 27.3 m in diameter (range 22.5 to 20.0 m). Sporangia were produced from CPA plugs flooded with 15% soil extract solution and were oblong to ovoid to ellipsoid in shape and semi-papillate (Fig. 3B). Sporangia averaged 67.5 m in length (range 37 to 87 m) and 29 m in width (range 25 to 35 m), with an average length to breadth ratio of 2.3 (range 1.5 to 2.8). Phytophthora spp. were not detected in any of the soil samples by baiting and they were not recovered from root pieces on PARP medium.



A
 
B

Fig. 3. Sexual and asexual propagules of Phytophthora siskiyouensis: (A) developing oospore of P. siskiyouensis with a paragynous antheridium; (B) ellipsoid, semi-papillate sporangia, including one sporangium releasing zoospores (inset).


Isolates obtained from single zoospores (Fig. 3B inset) were used for rDNA sequence analysis and pathogenicity experiments. Genomic DNA was extracted from Phytophthora cultures using the Qiagen DNeasy Kit (Qiagen, Valencia, CA) and the intergenic transcribed spacer (ITS) region of rDNA was amplified using primers ITS1 and ITS4 (9). The rDNA sequence of our isolate, GenBank no. EU818783, matched (with 100% identity) sequences (GenBank nos. EF490682, EF490683, EF490684, EF523386, EF523387) which were from a recently described species of Phytophthora, P. siskiyouensis Reeser and E.M Hansen (3,4,5). The alder isolates also matched the morphological description of P. siskiyouensis. Two isolates of P. siskiyouensis from alder trees in California have been deposited at the Centraalbureau voor Schimmelcultures collection (CBS122205, CBS122206).

Pathogenicity experiments were conducted using one isolate of P. siskiyouensis and three species of alder grown in California: Italian alder (Alnus cordata), white alder (A. rhombifolia), and red alder (A. rubra). White alder trees were purchased in containers from a local nursery, and Italian and red alder trees were purchased as dormant bare-root plants; they were potted and forced from dormancy in a greenhouse. Red and white alder plants were 1-year-old seedlings and Italian alder plants were 2-year-old seedlings. All plants were inoculated by placing CPA plugs with mycelia of P. siskiyouensis in small wedge-shaped wounds (approximately 1 cm) made through the bark into the cambium at the base of each tree stem. Control trees received only carrot piece agar plugs. All wounds were wrapped with parafilm for 48 h to prevent desiccation and encourage infection. Five trees of each species were inoculated, with an equal number of trees with agar plug controls. Trees were grown at 18C with a 12-h-photoperiod in a growth chamber for 3 weeks. Lesion length was measured as the sum of the distances the necrosis extended apically and distally from the point of inoculation. Small pieces from the edge of the lesion from each tree were placed onto PARP medium. Plates were monitored for growth for 14 days. The experiment was repeated once.

Three weeks after inoculation, trees of all three alder species inoculated with P. siskiyouensis had black, sunken lesions at the points of inoculation (Figs. 4A left, 4B left, and 4C left). Some of the inoculated alder trees exhibited black vascular streaking that extended beyond the lesions and into the roots and upper stems (Fig. 4B left). Italian alder trees had a lesion length average and standard deviation of 37.3  16.5 mm; whereas red and white alder trees had lesion averages and standard deviations of 23.6  5.5 mm and 21.0  9.3 mm respectively (Fig. 5). Phytophthora siskiyouensis was isolated from lesions on all inoculated trees. Control trees exhibited no lesions or discoloration, and P. siskiyouensis was not isolated from these trees (Figs. 4A right, 4B right, and 4C right).



A

  Fig. 4. Symptoms on stems of three alder species that were wounded and inoculated with P. siskiyouensis (in carrot piece agar plugs) or not inoculated:

(AAlnus rhombifolia, inoculated (left) and not inoculated (right);














(BAlnus rubra, inoculated (left) and not inoculated (right);














(CAlnus cordata, inoculated (left) and not inoculated (right).



 

B

 

C
 

 

Fig. 5. Mean lesion lengths on stems of three species of alder trees that were wounded and inoculated with P. siskiyouensis 3 weeks after inoculation; bars represent one standard deviation.

 

Summary

Phytophthora siskiyouensis was isolated consistently from dying Italian alder trees in a commercial landscape in Foster City, CA. In a growth chamber pathogenicity experiment, an isolate of P. siskiyouensis was confirmed to be pathogenic on Italian alder trees and was demonstrated to be potentially pathogenic to red and white alder trees. Phytophthora siskiyouensis caused black, sunken lesions on wounded stems only 3 weeks after inoculation, and was isolated from lesions on all inoculated trees. It is not known whether P. siskiyouensis is endemic to California or if it was introduced. It was suggested that P. siskiyouensis is a natural component of the mycobiota of the forests in southwestern Oregon (3). To date, this is the only location in California where P. siskiyouensis has been found.

Foster City is located in an area of coastal California adjacent to San Francisco Bay and approximately 19.3 km east of the Pacific Ocean. Climatic conditions of Foster City, CA, are similar to those of coastal Oregon where P. siskiyouensis was first detected while monitoring streams and soils for P. ramorum using leaves of rhododendron and tanoak as baits (3). Although infrequent, P. siskiyouensis has been isolated from bole cankers of tanoak and a blighted shoot of California bay laurel in Oregon. Phytophthora siskiyouensis also has been isolated elsewhere from dying alder trees (Alnus glutinosa) in a garden in Melbourne, Australia (7). The climate in this region is similar to that of Foster City and southwest Oregon. Although P. siskiyouensis was detected from soil in Oregon forests (3), it has not been found in soil around diseased alder trees. In this study, we did not recover P. siskiyouensis from soil beneath symptomatic trees by baiting with pear fruits. In Australia, it was not detected from soil from around diseased alder trees using pear fruits and eucalyptus cotyledons as baits (7). Perhaps P. siskiyouensis is more likely to be detected from soil using leaves of Rhododendron sp. or Chaemaecyparis lawsoniana as baits (3). Alternatively, January might not have been a good month of the year to sample soil for the presence of P. siskiyouensis in California.

Irrigation practices at the site in Foster City may have played a role in the disease development. Infective activity of Phytophthora spp. is favored by high moisture, heavy irrigation, and waterlogged soils (1). Sprinkler emitters were present near most alder trees at the site. Many of the emitters were adjacent to and pointing directly at the trunks. Although irrigation to those trees had ceased, improper watering practices when the trees were young may have contributed to initiation of disease at the site. Most of the soils in Foster City, CA, have poor drainage due to a high water table, 76 to 244 cm depending on season, precipitation and specific soil conditions, further favoring disease development by Phytophthora spp.

Little is known about the host range of P. siskiyouensis. Besides Alnus glutinosa, Phytophthora siskiyouensis was shown to be a potential weak pathogen on seedlings of other tree species, including Citrus, Acacia, and Eucalyptus (7). Previously it has been demonstrated that P. siskiyouensis is capable of causing substantial lesions on artificially inoculated tanoak stems in 4 weeks (4). All of the Italian alder trees at the Foster City site have been removed because of concern that a potentially exotic species of Phytophthora may become established in California and threaten the alder tree population. To date, replacement trees have not been planted at the site. This is the first report of P. siskiyouensis in California and the first report of this species causing collar rot on Italian alder trees, Alnus cordata.


Literature Cited

1. Erwin, D. C., and Ribeiro, O. K. 1996. Phytophthora Diseases Worldwide. American Phytopathological Society, St Paul, MN.

2. Jeffers, S. N., and Martin, S. B. 1986. Comparison of two media selective for Phytophthora and Pythium species. Plant Dis. 70:1038-1043.

3. Reeser, P. W., Hansen, E. M., and Sutton, W. 2007. Phytophthora siskiyouensis, a new species from soil, water, myrtlewood (Umbellularia californica) and tanoak (Lithocarpus densiflorus) in southwest Oregon. Mycologia 99:639-643.

4. Reeser, P. W., Sutton, W., and Hansen, E. M. 2008. Phytophthora species causing tanoak stem cankers in southwestern Oregon. Plant Dis. 92:1252.

5. Rooney-Latham, S., Blomquist, C. L., Pastalka, T., and Costello, L. 2007. First report of Phytophthora siskiyouensis causing disease on Italian alder in Foster City, CA. Phytopathology 97:S101.

6. Sinclair, W. A., and Lyon, H. H. 2005. Diseases of Trees and Shrubs, 2nd Edn. Cornell Univ. Press, Ithaca, NY.

7. Smith, I. A., Cunnington, J., and Pascoe, I. 2004. Another new? species of Phytophthora on alder down under (Australia). P. of the IUFRO Res. Work. Gr. Conf. on Phytophthora Forests and Natural Ecosystems. 11-17 September 2004. Freising, Germany.

8. Werres, S., Marwitz, R., Poerschke, U., and Themann, K. 2001. A long-term study of Phytophthora species in Germany. I Phytophthora species which could be definitely identified. Z. Pflanzenk. Pflanzen. 108:113-120.

9. White, T. J., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315-321 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innins, D. H. Gelfand, J. J. Sninsky, T. J. White, eds. Academic Press, San Diego, CA.