Pathogenicity of Leptographium serpens to Longleaf Pine Matusick, G., Eckhardt, L.G., Enebak, S.A. School of Forestry and Wildlife Sciences, Auburn University ABSTRACT Decline and mortality syndromes of southern pine species have attracted an increasing amount of attention from land managers over the past decade. The southern pine decline disease syndrome is characterized by complex interactions between several biotic and abiotic factors including root inhabiting fungi from the genera Leptographium, Grosmannia and Ophiostoma. Leptographium serpens is known to be associated with mortality of southern pines as part of the decline syndrome. The importance of longleaf pine to the southern pine ecosystem has been realized by many as its native range has decreased over the past century. Longleaf pines with roots colonized by either L. serpens or G. serpens have shown wilting symptoms preceding the death of the healthy foliage (Figure 1). Below-ground symptoms are more apparent and often include resinous lesions occluding dead roots, a distinct streaking through the root flair, and a deterioration of fine root mass. A more direct measure of pathogenicity of this pathogen on longleaf pine will establish the exact relationship between pathogen and host. The objectives of the project include the measurement of pathogenicity of L. serpens in relation to longleaf pine seedlings and mature roots. MATERIALS AND METHODS Seedling Inoculation Four hundred and eighty bare root longleaf seedlings were planted shortly after lifting in January of 2006. The seedlings were distributed evenly among 12 permanently placed above ground boxes (Figure 2, Figure 3). Two weeks after planting, 20 seedlings were wound inoculated in the lower stem (adapted from Nevill et al., 1995) within each box (Figure 4). Each seedling was inoculated with L. serpens ( isolate LOB-R-03-1606), which was isolated from the lateral root of a loblolly pine in Alabama. Also within each box, 10 seedlings were wounded and 10 were unwounded controls. In addition to inoculation, seedlings were exposed to one of three soil moisture levels (high, low, and normal). Four boxes were randomly assigned to the high (20%), low (5%), and normal (10%) moisture levels (Figure 2). The seedlings were allowed to grow for four months after which were removed from the boxes and seedling characteristics were measured. Survival, bud break, lesion presence and length as well as occlusion presence and length, biomass, and root collar diameter were variables of interest. Occlusion measurements were measured by aid of a Fast Green stain solution (adapted from Parmeter et al., 1989). Statistical analysis was conducted using the SAS software (SAS Institute, 9th ed., Cary, NC). Figure 3: Seedling inoculation setup, forty longleaf pine seedlings per box. Figure 4: Longleaf seedling wound inoculation in the lower stem. Lower Stem Wound Inoculation Figure 2: Seedling inoculation setup, twelve boxes divided between three different soil moisture treatments. Figure 8: Loblolly 10-20 age class mature tree inoculation site Figure 6: Loblolly lateral root inoculation with L. serpens RESULTS Seedling inoculation (Year 1) A total of one hundred and thirty-nine seedlings died over the four month study. Of those that died, 58% were inoculated with L. serpens, more importantly 33% of all inoculated seedlings died. Mortality within the inoculated treatment was significantly larger than that of the control treatment (Figure 9). Figure 9: Seedling mortality by treatment DISCUSSION Results from the seedling inoculation studies indicate Leptographium serpens is pathogenic to longleaf pine seedlings. Lesions were produced on all inoculated seedlings, with eighty one (33%) dying over the course of the study. Mortality rates were significantly higher within the wound inoculation treatment compared to the non-wounded control (Figure 9). In similar inoculation studies involving loblolly pine, mortality was not significant (Eckhardt et al., 2004; Nevill et al., 1995). This discrepancy in findings may suggest differences in host seedling vulnerability to Leptographium species. In addition to survival, absence or presence of bud break may indicate seedling health. The occurrence of bud break was significantly lower in inoculated seedlings when compared to the other two treatments (Figure 10). The presence of L. serpens may have imposed a significant stress on the seedlings, preventing emergence of the bud. Manipulation of the soil moisture levels allowed us to simulate both flood and drought conditions. It is known that abiotic factors contribute to decline disease systems (Manion, 1981). In the southeastern Unites States, pines, particularly those planted off site, experience both high and low soil moisture levels. Longleaf pine is known to naturally occur predominately on ridge tops in the coastal plain. Its enlarged tap-root makes it relatively drought tolerant and suggests it has evolved in an area of low soil moisture. Therefore, we would expect longleaf seedlings to be more tolerant of the low soil moisture level, as compared to the high level. Although, mortality was not significant between moisture levels, presence of occlusion was significant. Inoculated seedlings grown in the high moisture levels were more likely to produce occlusions in the stem, as well as longer occlusions (Figure 11). These data suggest host response to pathogen invasion may be affected by stresses such as high moisture content. Figure 10: Percent of seedlings with bud break Occlusion was restricted to the inoculated seedlings. Eighty-six percent of wound inoculated seedlings grown in the high soil moisture level had occlusions. Only fifty-one percent of wound inoculated seedlings exposed to the dry and normal moisture levels were occluded. Of the wound inoculated seedlings, occurrence of occlusion was more prominent within the wet soil treatment (Figure 11). Figure 11: Occlusion of wound inoculated seedlings by moisture INTRODUCTION The decline disease of longleaf pine (Pinus palustris) has attracted a considerable amount of attention in recent years due to changing management practices throughout the southern U.S. Restoration of historical longleaf habitat, along with production of timber for longleaf poles has become the main focus of some land managers. Extensive longleaf mortality has been observed in the 30-45 year old age class (Otrosina et al., 1999) as well as others (Eckhardt unpublished). Longleaf pine is capable of living for nearly 500 years. Unfortunately, old-growth longleaf pine individuals are not being replaced due to death of the younger age classes. Decline diseases are characterized by a combination of biotic and abiotic stresses (Manion, 1981). Several abiotic stressors that may influence longleaf pine health include, fire, wind, and moisture. The confirmation that Leptographium and Grosmannia species are the main biotic fungal causal agents in the decline of loblolly pine (P. taeda), (Eckhardt, 2003) indicates they may be involved with longleaf pine decline. Several Leptographium and Grosmannia species have been isolated from longleaf pine trees exhibiting decline symptomology (Otrosina et al., 1999; Otrosina et al., 2002; Zanzot and Eckhardt unpublished); these symptoms include yellowing, wilting foliage, low crown density, and premature needle drop. Some Leptographium and Grosmannia spp isolated from roots include, L. procerum, L. terebrantis, L. huntii, and L. serpens. Recently, Leptgraphium serpens has also been isolated from longleaf pines displaying crown symptoms (Eckhardt and Menard unpublished). In pathogenicity experiments involving four Leptographium species commonly recovered from loblolly pine seedlings, L. serpens produced the largest lesion area and second longest mean lesion length (Eckhardt et al., 2004). Despite its apparent virulence on loblolly pine, its pathogenicity on longleaf pine has not been established. These pathogenicity experiments will determine the relationship L. serpens has with longleaf pine and its role in longleaf pine mortality. Mature Tree Inoculation A series of mature tree inoculations will be conducted to determine the relative pathogenicity of four commonly isolated Leptographium species to two southern pine hosts. Leptographium procerum, L. terebrantis, L. huntii, and L. serpens isolates will be used in root inoculations of both longleaf and loblolly pine (P. taeda) trees over a range of age classes. Fifty-four trees will be inoculated in each of the following age classes: Longleaf Pine: 20-30, 40+; Loblolly Pine: 10-20, 20- 30, 30-40. One set of inoculations will be conducted on each tree species and age class each fall and spring, starting in the fall of 2006 and finishing in the spring of 2008. Inoculations conducted in the fall and spring coincide with peak insect vector populations. This experiment should establish the relative pathogenicity of the four Leptographium species (L. procerum, L. terebrantis, L. huntii, L. serpens) to each of the two tree hosts, as well as investigate any seasonal changes in host response. Two lateral roots on each tree will be excavated approximately three feet from the root collar (Figure 5). Each tree will receive a random combination of two Leptographium species, one fungal species per root. The inoculation will take place two feet from the root collar (Figure 6). Either a wound or sterile media control will be placed one foot from the root collar (Figure 7). Inoculated roots will be flagged, reburied, and left undisturbed for approximately sixty days (Figure 8). Roots will be re-excavated and removed for measurements. The diameter of the root, along with length, depth, and width of lesion will be measured. Figure 5: Loblolly tree with two lateral root inoculations 1 ft 2 ft 1 ft 2 ft L. terebrantis W. Control L. huntii M. Control Figure 7: Mature tree root inoculation setup Average Seedling Mortality between Treatments Wounded Non-Wounded Control Wound Inoculated 0 5 10 15 20 25 30 35 40 Treatments Seedling Mortality % Lesion occurrence was significantly greater on wound inoculated seedlings compared to wounded and non-wounded control seedlings. The mean lesion and occlusion lengths for wound inoculated seedlings were 17.6 mm and 19.9 mm respectively. Seedling shoot biomass, root biomass, and root collar diameter did not differ among the three treatments. Six percent of living inoculated seedlings had broken bud compared to 41% and 31% for control and wounded treatments, respectively. The percent bud break of inoculated seedlings was significantly less than the other two treatments (Figure 10). Percent of Seedlings with Bud Break Wounded Non-Wounded Control Wound Inoculated 0 5 10 15 20 25 30 35 40 45 50 Treatments Percent Bud Break % Percent Occlusion of Wound Inoculated Seedlings between Moisture Levels Dry Wet Normal 0 20 40 60 80 100 Moisture Level Percent Occlusion REFERENCES 1. Eckhardt, L.G., Jones, J.P., Klepzig, K.D. 2004. Pathogenicity of Leptographium Species Associated with Loblolly Pine Decline. Plant Disease 88: 1174-1178. 2. Eckhardt, L.G. 2003. Biology and Ecology of Leptographium Species and Their Vectors as Components of Loblolly Pine Decline. PhD. Diss. Louisiana State University, Baton Rouge. 3. Manion, P.D., 1981. Tree Disease Concepts, Prentice Hall, Englewood Cliffs, NJ 402 pp. 4. Nevill, R.J., Kelley, W.D., Hess, N.J., Perry, T.J. 1995. Pathogenicity to loblolly pines of fungi recovered from trees attacked by southern pine beetles. South. J. Appl. For. 19: 78-83. 5. Otrosina, W.J., Bannwart, D., Roncadori,R.W. 1999. Root-infecting fungi associated with a decline of longleaf pine in the southeastern United States. Plant and Soil 217: 145-150. 6. Otrosina, W.J., Walkinshaw, S.H., Zarnoch, S.J., Sung, Shi-Jean, Sullivan, B.T. 2002. Root Disease, Longleaf Pine Mortality, and Prescribed Burning. Proceedings from the eleventh biennial southern silivicultural research conference 7. Paremeter, J R., Slaughter, G.W., Chen, M.M., Wood, D.L., Stubbs, H.A. 1989. Single and mixed inoculations of Ponderosa pine with fungal associated of Dendroctonus spp. Phytopathology 79: 768-772. A A A B B A Southern Forest Nursery Management Cooperative Figure 1: Declining trees in a longleaf forest AB A B A A For More Information Contact: George Matusick