Decline and Death of Pinus Spp. in Delaware Caused by Bursaphelenchus Xylophilus ~ J

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Decline and Death of Pinus spp. in Delaware Caused by Bursaphelenchus xylophilus ~ J. C. ADAMS AND A. L. ~{OREHART2 Abstract: Etiological studies to determine the cause of decline and death of Pinus spp. in Delaware were initiated in 1980. The pinewood nematode, Bursaphelenchus xylophilus, was found to be the major canse of mortality in Japanese black pine (Pinus thunbergii). "When inoculated into health,,' 5-yr-old Japanese black pines, B. xylophihts produced typical decline syntptonls observed in the lieht. The xylophilous fungi nit)st often associated with declining trees, Rhizosphaera pint, Fttsarium spp., and Pestalotia Junerea, were not pathogenic to Jap- anese black pine in greenhouse tests. Mineral analyses of soil and foliage showed no significant differences between healthy and infested trees. B. xylolyhilus was also found on loblolly pine (P. taeda), scrub pine (P. virginiana), Scots pine (P. sylvestris), red pine (P. resbmsa), Eastern white pine (P. strobus), and pitch pine (P. rigida). Key words: pinewood nematode, Japanese black pine. ,Journal of Nematology 14(3):382-385. 1982.

Bursaphelenchus xylophilus (Steiner MATERIALS AND METHODS and Buhrer 1934) Nickle 1970 (syn. B. lignicolus, Manfiya and Kiyohara 1972), the To determine the incidence of pinewood pinewood nematode, induces a rapid decline nematode, random collections were made in and death of several Pinus species and other New Castle and Sussex counties from symp- conifers (1,13). The nematode is a problem tomatic pines during a 6-wk period from in Japanese forests as evidenced by epidem- January to February 1981. Several branches ics on Japanese black and Japanese red were removed at breast height from each pines (P. thunbergii Parl. and P. densiflora tree. On Japanese black pine sites, needles Sieb. & Zucc., respectively) (5,9). Japanese and soil samples from symptomatic and black pine is planted widely along Delaware asyznptomatic trees were taken for study. beach areas because it stabilizes dunes, is No samples were taken in Kent County aesthetically attractive, and is tolerant of because of the limited distribution of Pinus the coastal environment. D~cline and death spp. in that part of Delaware (4). of Japanese black pine has been observed Bursaphelenchus xylophilus was ex- for 10 yr in Delaware and was generally tracted from the samples by the Baermann attributed to poor species adaptation (R. B. funnel technique (3). After removing the Carroll and A. L. Morehart personal com- bark, branches were cut into ca. 5-cln ~' muncation). Repeated isolations and tissue pieces. Three replicate 100-cm 3 samples analyses failed to yield phytopathogenic were placed in distilled water in plastic agents or fertility problems. Reports of B. funnels and left overnight. To quantify xylophilus in several states (13) prompted populations, triplicate 3-ml aliquots were a further evaluation of the problem in taken from each replicate and nematodes Delaware. The pinewood nematode was were counted under a dissecting microscope. found on Japanese black pine in the fall of Pathogenicity of B. xylophilus recovered 1980, and the identification was confirmed from survey samples was tested using 5-yr- by Dr. Lorin Krnsberg, University of Mary- old Japanese black pines growing at New- land, College Park (L. Krusberg personal ark, Delaware. Nematodes were extracted as communication). This report summarizes a described and centrifuged in distilled water survey designed to determine the incidence at 1,000 × g to concentrate inoculum. A and severity of pinewood nematode in Del- 1.0-ml-capaci ty syringe fitted with a 26-gauge aware. needle was used to introduce ca. 10 a nematodes/tree into terminal buds of heahhy trees. The wounded area was then sealed Received for publication 8 I)ecember 1981. with Parafilm. Control trees were similarly ~This research was supported by funds from the McSntire- Stenni.s Cooperative Forestry Research Program, inoculated with distilled water. Trees were ~Graduate Fellow a:ld Professor. ])('partnn'nt of Plant harvested 6 wk after inoculation, divided in Science, University of Delaware, New,ark I)E 197tl. Pub- lished with the approval of the Direttor of the Delaware 5-cm sections, and nematode determinations Agriculnlral Experiment Station as Miscellatieous No. 965. were made, as previously described. Contribution No. 132 of tile Deparlment of l'lant Science, University of Delaware, Newark DE 19711. To determine the presence of pathogenic 382 Bursaphelenchus xylophilus: Adams, Morehart 383 and/or associated micro-organisms, small chips of wood from each sample were surface disinfested in 10% clorox v/v (0.5% sodium hypochlorite) followed by 70% ethanol v/v, rinsed in sterile distilled water, and incubated in tile dark at 28 ± 1 C on acidified potato dextrose agar. Fungi most frequently recovered (Pestalotia [unerea, Fusarium sp., anti Rhizosphaera pini) were tested individually for pathogenicity to 8- wk-old Japanese black pines in the greenhouse. In the first test, fungal inoculum was introduced into steam-pasteurized soil, followed by planting of pine seedlings. Inoe- ulum was prepared from 2-wk-old fungal cultures grown on potato dextrose agar at 28 __+ 1 C. Four cultures were mixed with 400 ml sterile distilled water in a Waring Blendor, and 100 ml of inoculum was hand mixed into the soil of each replicate. Con- trol soil was treated with an identical volume of an agar slurry. In the second test, pine seedlings growing in pasteurized soil were wounded at the base of the stem with a sterile scalpel and agar plugs of fungal mycelium were placed in the wound, which was then wrapped with Parafilm. Control trees were wounded and inoculated only with agar plugs. Both experiments were + complete randomized block designs with A

six replications per treatment. Seedlings + were observed for symptom development for SUSSEX 12 wk, then harvested, disinfested, and incubated at 28 ~ 1 C on acidified potato dex- + trose agar.

Dilutions of soil samples from Japanese 4- black pine sites were performed as described by Johnson and Curl (6), using dilutions of Fig. 1. Distribution in Delaware of Pinus spp. in- l0 -~ and l0 -~ on acidified potato dextrose fected with Bursaphelenchus xylophitus. agar and OAES medium (17). Plant tissue and sixty-nine percent of 13 symptomatic and soil mineral analyses were performed loblolly pines examined were positive for by the Soil Testing Laboratory, University B. xlyophilus. All samples from other spe- of Delaware, Newark. cies (sample numbers were 5, 12, 5, 3, and 8 RESULTS for P. resinosa, P. sylvestris, P. virginiana, P. strobus, and P. rigida, respectively) were Bursaphelenchus xylophilus was found positive. Nematodes extracted from Jap- in tissue of seven species of pine in Del- anese black pine, inoculated into terminal aware: red (P. resinosa Ait.,), Scots (P. buds of healthy Japanese black pines grow- sylvestris L.), scrub (P. virginiana Mill.), ing in Newark, Delaware, produced decline Eastern white (P. strobus L.), loblolly (P. symptoms (severe chlorosis followed by taeda L.), pitch (P. rigida Mill.), and Jap- needle necrosis) within 3 wk. Although anese black (P. thunbergii). Distribution symptoms were severe, death did not occur of these species is ontlined in Fig. 1. Ninety- before harvest. Large populations of B. seven percent of 67 Japanese black pines xylophilus were recovered from these trees 384 Journal of Nematology, Volume 14, No. 3, July 1982 several centimeters below the point of in- isolated, appearing in 80.0, 66.1, and 44.6 % oculation. No nematodes were recovered of the samples, respectively. Isolates of these from controls. three fungi were not pathogenic when inoc- Population levels of B. xylophilus were ulated into soil around roots or into stems compared between loblolly, .Japanese black, of 8-wk-old Japanese black pines growing Scots, and pitch pine. Populations of nema- in the greenhouse. Dilutions of soil samples todes from Japanese black pine were sig- from Japanese black pine sites failed to nificantly higher (P = 0.05) than other yield any phytopathogenic fungi, with the species, none of which differed significantly exception of one site, which was negative (Table 1). for the presence of B. xylophilus. Textural and nutritional analyses of Table 1. Estimated populations of Bursaphel- soils indicated that soil factors have little enchus xylophilus on declining Pinus spp.* or no effect on nematode infestation of Japanese black pine. A large variation ex- Species No. of B. xylophilus S.D. isted in elemental and organic matter con- tent for both infested and noninfested sites. P. thunbergii 112.8 A'~ +27.6 Tissue analysis of Japanese black pine foli- P. radiata 28.0 B + 5.8 age on the same sites yielded no significant P. sylvestris 29.5 B ± 10.7 P. taeda 31.5 B ±11.8 differences in element concentrations between infested and noninfested trees. *Populations are expressed as the mean of three 3-ml aliquots taken from a Baermann funnel determination of a 100-cm3 sample. DISCUSSION "~Means followed I)y the same letter are not sig- Of 18 pine species listed as hosts of B. nificantly different (P = 0.05) according to Duncan's multiple-range test. xylophihts in tile U.S. (13), 16 are from the subgenus Diploxylon (12); six of seven Several xylophilous fungi were found host species found in Delaware were associated with nematode-infested Japanese Diploxylon pines. It is not immediately black pine (Table 2). These included evident whether this indicates a greater Botrytis cinerea, Ceratocystis sp., Clado- susceptibility of two- and three-needle pines, sporium sp., Fusarium spp., Gliocladium reflects the greater number and distribution roseum, Pestalotia [unerea, Rhizoctonia of these pine species, or results from spe- solani, and Rhizosphaera pini. Of these cificity in feeding habits of the vector fungi, Pestalotia funerea, Fusarium spp., Cerambycid beetles. Our results, based on and Rhizosphaera pini were most frequently population levels of the nematode, suggest a Table 2. Xylophilic fungi associated with difference in susceptibility among Diploxy- Bursaphelenchus xylophihts-infested Pinus thun- Ion species. Japanese black pine consistently bergii. yielded higher nematode populations than the other three species tested, all of which Sample were Diploxylon (Table 1). Suzuki and Species Frequency* Percentage* Kiyohara (15) observed that small initial B. xylophilus populations (one male and Bot~ytis cinerea 1.3 7.7 one virgin female) inoculated into Jap- Ceratocystis sp. 1.8 4.6 anese black pine seedlings produced large Cladosporium sp. 0.9 7.7 Fusarium spp. 4.9 66.1 nematode numbers in one month. The same Gliocladium roseum 2.7 16.9 authors (7) also reported a guantitative re- Pestalotia funerea 8.8 80.0 lationship between nematode number and Rhizoctonia solani 0.6 4.6 symptom development in affected pines. Rhizosphaera pini 3.9 44.6 The possibility, however, of greater frequency in feeding by the vector (hence *Percent isolation based on a total of 1,040 at- large initial B. xylophilus populations) on tempts. ~Percent isolation from 65 samples (100% = oc- Japanese black pines should not be ex- currence on at least one isolation from all 65 cluded. We are presently using greenhouse samples). and laboratory screeing methods to deter- Bursaphelenchus xylophilus: Adams, Morehart ~85 mine if field observations can be reproduced observed in the beginning of our survey witlt controlled inoculum levels. were a result of drougfit-induced predisposi- Our observations of fungi associated tion of Pintts spp. during the 1980 growing with B. xylophilus infestations are similar season. to those of previous reports (8,10). Several LITERATURE CITED of tile fungi isolated have been reported as pathogens of Japanese black pine (14), but 1. Carling, D. E., R. C. Lambe, S. A. Alexander, are probably not involved in the etiology of and A, E. Elliot. 1981. Pinewood nematode disease in ornamental pines in Virginia, Phytopathology pine decline, based on greenhouse patlto- 71:765. genicity tests. Peslalotia [unerea, Fusarium 2. Commerce Department. 1980-1981. Climatolog- spp., and Rhizosphaera pint were isolated ical data: Maryland and Delaware. Vol. 84-85. Natl. frequently, but are typically associated with ('limatic Cent., Asheville, N, C. secontlary invasion of diseased tissue (16). 3. Dropkin, V. H. 1980. Introduction to plant uematology. New York: John Wiley & Sons. Species in the genus Bursaphelenchus are 4. Ferguson. R. H., and C. -~V. Mayer. 1973. The ntycophagous (11); we often observed feed- timber resources of Delaware. USDA, Forest Service, lug activity of nematodes on mycelium from Res. Bull. NE-32. surface-disinfested wood chips, particularly 5. Ishibashi, N., and E. Kondo, 1977. Occurrence aml siJrvival of the dispersal forms of pinewood with Gliocladium roseum and Rhizosphaera nematode, Bursaphelenchus lignicolus Mamiya and pint. These fungi could possibly enhance Kiyohara. Appl. Entom. Zool. 12:293-2~02. nematode survival in drying tissue. 6. Johnson, L. F., and E, A. Curl. 1972. Methods Inconclusive results from soil and tissue for research on the ecology of soil-bnrne plant pathogens, Minneapolis: Burgess Publishing Co. analyses were expected, considering tile wide 7. Kiyohara, T., and K. Suzuki, 1978, Nematode distribution of B. xylophilus on several population growth and disease development in the species growing in a variety of soil types. We pine willing disease. Eur. J. For. Pathol. 8:285-292. did not include a nitrogen analysis in our 8. Kobayashi, T., K. Sasaki, antt Y. Mamiya. study because previous work showed no 1975, Fungi associated with Bnrsaphelenchus lig- uicolus, the pinewood nematode (1). J. Jap. For. correlation between nitrogen levels and de- Soc. 56:136-145. cline incidence (R. B. Carroll personal com- 9, Mamiya. Y.. and T. Kiyohara. 1972. 1)escrip- munication). tion of Bursaphelenchns lignieolus n. sp. (Nema- Pinewood nematode has reached epi- toda: Aphelenchoididae) from pinewood and histo- pathology of nematode-infested trees. Nematologica demic proportions on Japanese black pine 18:120-124. in several parts of southern Delaware. Using 10. McGawley, E. C., J. P. Jones, and W. Birch- color and false-color infrared aerial photo- lield. 1980. Reproduction of Bursaphelenchus lig- graphy, verified by ground-truth studies, uicolns on fungi isolated from B. lignicolus infested pine trees. ,l, Nematology 12:231 (Abstr.), we have estimated a 35% incidence of B. i l. Nickle, W. R. 1970. A taxonomic review of x),lophilus on Pinus spp. at Cape Henlopen the genera of lhe Aphelenchoidea (Fncbs, 1937) State Park, in Sussex County. Scots pines Thurue, 1949 (Nematoda: Tylenchida). J. Nema- growing in northern New Castle County tology 2:375-392. have sustained equal damage in 15-25-yr- 12. Pelerson, R. 1980. The pine tree book. New York: Brandywine Press. okl stands. 13. Rnbbins, Kathryn. 1981. Known distribution We have not, however, observed any of Bursaphelenchus lignicolus in the United States/ significant increase in the incidence of B. March 1981. Northeastern Area, State and Private Forestry, USDA, Forest Service. xylophilus from May to August I981. Ac- 14. Spaulding, P. 1961. Foreign diseases of forest cording to U.S. Department of Commerce trees of the world. Agr. Handbook No. 197, USDA, data for 1980 and 198I (2), mean tempera- Forest Service. tures were lower, and total precipitation 15. Suznki, K., and T. Kiyohara. 1978. Influence of water stress on development of pine wilting greater, during May-August 1981, com- disease caused by Bursaphelenchus lignicohls. Enr. pared to tile same period in 1980. Studies J. For. Pathol. 8:97-107. (7,15) have shown that water stress greatly 16. Tarr, S..4,. J. 1972. The principles of plant increases symptom severity on nematode- pathology. New York: r~Vinchester Press. infested Japanese black pines. Possibly the 17. "Fnlte, J. 1969. Plant pathological methods: Fungi and hacteria. Minneapolis: Burgess Pnblishing high incidence of B. xylophiIus infestations Co.