Blue-stain Fungi Associated with Roots of Southern Pine Trees Attacked by the Southern Pine Beetle, Dendroctonus frontalis

Otrosina, W. J.. Hess, N. J., Zamoch. S. J.. Perry, T. J., and Jones, J. P 1997. Blue-stain fungi associated with roots of southem’pine trees attacked by the southern pine beetle, Dendmctonus front&s. Plant Dis. 81942945. Forty paired plots were established from eastern Texas to Alabama to study root-infecting, bluestain fungi in southern pine stands undergoing southern pine beetle (SPB) attack. Woody roots were sampled in plots undergoing recent or current attack by the SPB. Comparisons were made between occurrence of Lcptogrqhium spp. and related fungi and data on various characteristics of natural stands and plantations studied. Three fungal species, L terebrontis, L pnxerum, and Ophiostomo ips. along with unidentified kptographium and Grophium species, were isolated from sampled roots. L terebruntis was isolated more frequently from SPB-attacked plots (P c 0.001) than was either L. pmcerum or 0. ips. More blue-stain fungal species and related genera were isolated from SPB-attacked plots than from control plots (P < 0.001). This also was true for combined isolation percentages of L terebrantis, L. pmcerum, and 0. ips (P = 0.03). Presence of blue-stain fungi also was associated with higher stand basal area in the control plots (P = 0.045). Isolation frequencies of 0. ips and L procerum, along with the combination of these fungal species with L tercbrontis, were logistically related to increasing stand basal area in the control plots (P = 0.02. 0.02, and 0.01, respectively). No logistic relationship was found for frequency of any of the three blue-stain species with respect to basal area in SPB-attacked plots. These results suggest blue stain fungi are important in the dynamics of susceptibility of southern pines to SPB attack. There is considerable evidence linking the presence of root diseases and tree killing bark beetle attacks in conifers in the western United States (6,7.13.18). In the southeastern United States, the root-disease causing fungus, Hetembasidion amoston Fr. (Bref,), has been associated with susceptibility of loblolly pine (Pinus taeda L.) to attack by the southern pine beetle, Dendmctonus ftvntalis Zimm. (Coleoptera: Scolytidae) (1). Recent studies have revealed other root pathogens that are associated with trees exhibiting decline symptoms in plantations and natural stands (3,4.18). Root infecting fungi belonging to the genera Leptographium Lagerb. & Melin and Ophiostoma H.& P Sydow have been studied largely with respect to their relaCorresponding author: W. J. Otrosina 8-mail: [email protected] Accepted for publication 9 May 1997. Publication no. D-l 997-0902-01 R This article is in the public domain and not copy rightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1997. 942 Plant Disease / Vol. 81 No. 8 tionships with bark beetles (11). These fungi are commonly associated with bark beetles either as pathogens or as casual associates found in beetle galleries (9). Pathological relationships relative to their presence in roots and their role in predisposition of conifers to stem-attacking bark beetles have not been established for many of these fungi. This is especially true regarding the association of these fungi with pine roots and their possible role in processes involved in southern pine beetle (SPB) attack. Also, little is known of the distribution of Leptographiwn species in pine roots and their relationships to this economically and ecologically important bark beetle. The objectives of this study were to 1) determine the distribution and frequency of Leptographiutn species and related fungi in southern pine stands under SPB attack and 2) determine stand characteristics associated with occurrence of these root disease fungi. MATERIALS AND METHODS Forty paired, variable-radius plots were established in southern pine stands over a broad geographic area. Thirty-one of the 40 paired plots were located in loblolly pine stands, four were in slash pine (Pinus elliottii Engelm.) stands, four in mixed loblolly and slash pine stands, and one in a longleaf pine (Pinus pulustris Mill.) stand. The plot locations ranged from Alabama to Texas and represent a wide variety of southern pine sites. (Exact details of plot locations are available from the senior author.) Plot establishment was determined by locating SPB-attacked pine trees using the Southern Pine Beetle Information System (SPBIS), a computerized tracking system designed to identify and locate active beetle attack spots (12). The majority of these active SPB attack spots were located within 1 month of attack initiation. For each SPB plot, a control plot was established by traversing a line in a cardinal direction a distance of 80.5 m from the due north edge of the SPB plot. The control plots are presumed to represent stand and site conditions present in the SPB-attacked plots. In each of the control and SPB plots, data on tree species, diameter at breast height (DBH), pine basal area, tree height, radial growth increment, soil texture, aspect, stand basal area, and evidence of previous site disturbance were recorded. Trees used for determining DBH and radial growth measurements were obtained by selecting three dominant or codominant trees nearest the plot center. The height measurement used to obtain site index values for each SPB and control plot was measured by randomly selecting one of the three trees closest to the plot center. Roots from the three trees in each plot were sampled using a modified two-root excavation method (2). All root samples were taken within 30-40 days of initial attack dates recorded on SPBIS records. -0 primary lateral root segments greater than 3 cm in diameter from each of the three plot trees were excavated with hand tools to a distance of approximately 1 m from the root collar. Twenty-centimeterlong sections of rmt were cut beginning at 16 and 50 cm from the root collar from each root. The samples from each tree were placed in a plastic bag and kept chilled in ice chests no longer than 48 h before transportation to the laboratory. Two hundred and forty trees were sampled on the 80 plots in this study. Root samples were washed under nmning tap water, air dried, and cut into 1 cm x 0.5 cm sections. The wood sections were surface disinfected by immersing in 95% ethyl alcohol and flaming briefly. Three sections from the 16and SO-cm portions of each root segment from each tree were placed on each of three plates of malt extract agar (MEA), ortho-phenyl phenol medium (2), and cycloheximide amended malt extract medium (14). When staining or resinosis was observed on root samples, isolations were made at _ the margins of such features. Otherwise isolations were made from several points, along the root sample. Subcultures were, made from plates exhibiting fungal growth for identification and further study. The study design was a randomized block design in which each blocticonsisted of a SPB-attacked plot and a non-attacked control plot. Thus, the overall study had 40 blocks. Test of differences in stand characteristics between SPB and control plots used a standard randomized block analysis. Because there were only two treatments, the analysis was equivalent to a paired ttest. Analysis of presence or absence of blue-stain fungi was on a plot basis, where one confirmed fungal isolate from one tree in a plot was sufficient to score that plot positive. Differences in proportion of SPB and control plots with blue-stain fungi utilized the McNemar test (S), taking into account the blocking structure of the study. Logistic regression methods using PROC LOGIST (SAS Institute Inc., Cary, NC) were used to model the association between blue-stain fungi and stand conditions. RESULTS Cycloheximide-amended and unamended MEA were equally effective in isolating blue-stain fungi. The fungal species isolated from the root samples were 0. ips (Rumb.) C. Moreau, L terebrantis Barras & Perry, and L procerum (Kendr.) Wingfield. H. anmsum was not isolated from any root samples. Unidentified species of Leptographium and Graphium Corda isolated from root samples were included in certain analyses. The SPB plots sampled were either under current attack or were recently vacated (within 2 weeks of sample) as determined by presence of fresh pitch exudates or tubes, off-color foliage, and presence or absence of exit holes in the bark of attacked trees. Stand characteristics were similar between SPB-attacked and control plots (Table 1). Control and SPB plots within natural stands or plantations also had similar stand characteristics (Table 2). The proportion of blue-stain fungi isolated from plots in either plantations or natural stands was generally higher in the SPBinfested plots than in the control plots. However, only plots located in plantations had a significantly higher proportion of blue-stain fungi in SPB-attacked plots than in the control plots (Table 2). Control plots with blue-stain fungi, including those not identified to species, tended to have higher total basal area than did control plots from which no blue-stain fungi were isolated. SPB plots with blue-stain fungi had a higher lo-year growth increment than those SPB plots from which no blue-stain fungi were isolated (Table 3). When all three identified blue-stain species were pooled, a significantly higher frequency of occurrence of these fungi was present in the roots of SPB-infested plots (50%) relative to the control plots (25%) (Table 4). Overall, blue-stain fungal isolates, including the unidentified Leptographium and Graphium isolates, also had a higher frequency of occurrence in the SPB plots (87.5%) as compared to control plots (45%). Of the three identified bluestain species, only L.. terebrantis was pres‘hbie 1. Comparison of stand characteristics between control and southern pine beetle (SPB) paired