238 Florida Entomologist 96(1) March 2013

ROOT-INHABITING BARK (COLEOPTERA: ) AND THEIR FUNGAL ASSOCIATES BREEDING IN DYING LOBLOLLY PINE IN ALABAMA

George Matusick1,3, Roger D. Menard2, Yuan Zeng3 and Lori G. Eckhardt3 1The Nature Conservancy, Chattahoochie Fall Line Project, Fort Benning, GA, USA

2U.S. Department of Agriculture, Forest Service, Forest Health Protection. Pineville, LA, USA

3School of Forestry and Wildlife Sciences, Auburn University. Auburn, AL

*Corresponding author; E-mail: [email protected]

Root feeding bark beetles in the genus Hylastes breeding in loblolly pine across 3 crown (Coleoptera: Curculionidae: Scolytinae) common- condition classes (healthy, dying, dead). Addition- ly carry ophiostomatoid fungi (Ophistomatales: ally, the relationship between ophiostomatoid Ophistomataceae)and collectively contribute to species, their Hylastes species vectors, and tree root disorders of Pinus species around the world condition were investigated. (Jacobs & Wingfield 2001). One of the most dam- Loblolly pine stands across central Alabama aging root disorders is black-stain root disease have experienced high mortality rates, character- of conifers in the Western United States, caused ized by scattered stand mortality with a distinct by the fungal species Leptographium wageneri lack of above-ground pests (Brown & McDowell Kendrick and its primary vector H. nig- 1968). During an investigation of one stand in rinus (Mannerheim) (Witcosky et al. 1986). Re- May 2008 in central Alabama, dying trees were cently, Eichhoff and H. tenuis found to be infested with root-inhabiting beetles Eichhoff have been associated with loblolly pine and their associated ophiostomatoid fungi. Six (Pinus taeda L.) decline (Eckhardt et al. 2007), trees were selected based on crown condition, in- which is considered an emerging forest health is- cluding 2 with green, healthy crowns (healthy), sue in the southeastern United States (Eckhardt 2 with severely chlorotic and thinning crowns et al. 2010). (dying), and 2 with red crowns (dead). Two pri- Loblolly pine decline (LPD) is a tree disease mary lateral roots were randomly selected and complex that is characterized by symptoms that excavated to approximately 3 meters from the include thinning tree crowns, growth reductions root collar. Roots were removed from trees and (Eckhardt et al. 2007) and premature mortality in carefully dissected. All , including imma- localized areas (Brown and McDowell 1968). Evi- ture stages, were collected from roots and adult dence suggests the complex is the result of inter- beetles were identified to species. Root tissue was acting abiotic (Eckhardt & Menard 2008) and bi- obtained from each brood gallery for the isolation otic (Eckhardt et al. 2007) stress factors. Hylastes of ophiostomatoid fungal species using methods species vector a variety of root-infecting ophiosto- described in Eckhardt et al. (2007). Following matoid fungi (Klebzig et al. 1991, 1995; Eckhardt surface sterilization, tissue was placed on CSMA et al. 2007; Zanzot et al. 2010) which are patho- (malt extract agar containing 800 mg/L of cyclo- genic to loblolly pine (Matusick & Eckhardt 2010; heximide and 200 mg/l of streptomycin sulfate) Matusick et al. 2011). Hylastes beetles and their selective nutrient agar (Jacobs & Wingfield 2001). associated fungi are thought to collectively contrib- Each adult was also rolled on CSMA to re- ute to LPD by causing root damage through fungal cover ophiostomatoid fungi from insect bodies infection and insect feeding (Eckhardt et al. 2007). (Zanzot et al. 2010). Isolated ophiostomatoid fun- Root and lower stem feeding beetle species (Cole- gal species were identified using morphological optera: Curculionidae) including H. salebrosus and characters and established keys. Unknown iso- H. tenuis are found in increased numbers within lates were sequenced and confirmed as new spe- LPD-affected stands, compared to healthy stands cies by M. Wingfield (Forestry and Agricultural (Eckhardt et al. 2007, Menard 2007). A recent Biotechnology Institute, Pretoria, South Africa). study has found Hylastes to be the most common A total of 157 adult beetles (Coleoptera: Cur- genus observed in loblolly pine stands culionidae) were collected from roots, most from in central Alabama (Thompson 2011), illustrating dying trees (Table 1). A majority of the beetles col- their dominance in this ecosystem. Despite their lected were Hylastes species, including a total of dominance and potential for damage in loblolly 113 H. salebrosus (all from dying trees) and 34 pine, the breeding behavior of Hylastes species in H. tenuis (31 from dying and 3 from dead trees). the southeastern United States is not well under- In addition, H. salebrosus larvae and pupae were stood. In the current study, observational meth- collected from dying trees. Other adult insect spe- ods were used to investigate the potential for cies collected include the black turpentine beetle Scientific Notes 239

Table 1. The total number of beetles collected and isolated from healthy tree tissue, which supports beetles collected found transporting ophio- previous reports (Zanzot 2010). Leptographium stomatoid fungi in 6 loblolly pine trees repre- serpens, which was infrequently isolated from in- senting 3 health conditions. sects, was not isolated from root tissue. Addition- ally, one undescribed Ophiostoma species was in- Beetles frequently isolated from tissue during sampling. Tree Adult vectoring The findings reported here represent the first Tree Health Class # beetles (#) fungi (#) observations of Hylastes species breeding in roots Healthy1 1 0 0 of loblolly pine. Up until this point, the tissue used 2 0 0 for brood development of Hylastes species in the Dying2 3 26 19 southeastern United States has only been hypoth- 4 125 50 esized. These observations have implications on the LPD cycle by confirming dying trees contrib- Dead3 5 3 3 ute to increasing Hylastes populations, suggest- 6 3 2 ing a positive feedback cycle with regards to LPD in severely affected stands. These observations, Total 157 74 coupled with previous findings from elsewhere 1Sample trees with predominantly (>90%) healthy foliage (Witcosky & Hansen 1985) also suggest Hylastes (dark green). species development may be restricted to trees in 2Sample trees with predominantly (>70%) dying foliage (pale a dying condition. Additional research is needed green and yellow). to determine the root environmental conditions 3Sample trees lacking most (>90%) foliage, with the remain- ing being dead (red). required for Hylastes species development as well as to determine the potential for Hylastes species to invade and successfully reproduce in healthy (Dendroctonus terebrans (Olivier) (1-dying and trees, particularly where populations are high. 3-dead), and Xyleborus pubescens Zimmerman (6-dying trees). This examination is the first to Summary observe and provide evidence for Hylastes species breeding in loblolly pine roots and supports pre- Root feeding bark beetles in the family Cur- vious reports of Hylastes species breeding behav- culionidae and the ophiostomatoid fungi they ior in other, similar ecosystems (Blackman 1941; vector contribute to root disorders of Pinus Stoakley 1968; Witcosky & Hansen 1985). species around the world. In the southeastern Adult beetles were commonly found transport- United States root feeding beetles in the genus ing ophiostomatoid fungal species, including 47% Hylastes (Coleoptera: Curculionidae), including of all beetle species, 40% of H. salebrosus, and Hylastes salebrosus and , are as- 70% of H. tenuis. Collectively, insects transported sociated with stands of Pinus taeda experienc- a total of 5 ophiostomatoid species. Leptographi- ing premature mortality. Despite this, direct ev- um terebrantis was most commonly transported idence of Hylastes species breeding, with their (23% of all beetles). Additional species in order associated ophiostomatoid fungi, in Pinus taeda of frequency included, Grosmannia huntii (R.C. has not been observed until this point. In one Rob. Jeffr.) Zipfel, Z. W. de Beer & M. J. Wingfield P. taeda stand experiencing increased mortality (L. huntii M. J. Wingfield) (19%),L. procerum in Alabama, roots were excavated and dissected (6%), and L. serpens (Goid) M. J. Wingf. (1%). from pines exhibiting a range of crown condi- Seven insects transported more than one fungal tions in order to determine if Hylastes breeding species. In a similar loblolly pine stand in cen- and feeding damage could be detected and to tral Alabama, a large-scale trapping experiment investigate the relationship between beetles, found that H. salebrosus and H. tenuis transport- ophiostomatoid fungi, and tree crown condition. ed fungal species at slightly higher rates than Hylastes salebrosus and H. tenuis represented those reported here (Eckhardt et al. 2007). With the majority of beetles collected (94%) and were respect to the beetle-fungal association, data from found nearly exclusively in root of trees with this research supports conclusions made by Zan- dying crowns. Adult beetles and beetle galler- zot et al. (2010), that ophiostomatoid fungi are ies were associated with a host of ophiostoma- likely facultatively associated with Hylastes vec- toid fungi, including Leptographium terebranis, tors in the southeastern United States. Grosmannia huntii, L. procerum, and L. ser- Ophiostomatoid fungi were predominately iso- pens. These observations suggest roots of dying lated from dying and dead trees (Table 2). Lep- trees represent a valuable host substrate for tographium terebrantis and G. huntii were most expanding H. salebrosus and H. tenuis popula- frequently isolated including from root tissue tions. A more comprehensive study is required and beetles from both dead and dying trees. Lep- to confirm these findings. tographium procerum was infrequently isolated Key Words: galleries, Hylastes, loblolly pine from dying trees and was the only fungal species decline, ophiostomatoid fungi, root disorders 240 Florida Entomologist 96(1) March 2013

Table 2. Number of independent isolates of each of 4 ophiostomatoid fungi isolated from root tissue (beetle gal- leries) and adult beetle bodies, collected from loblolly pine trees within 3 health conditions.

1Dead Trees 2Dying Trees 3Healthy Trees

Fungal Species Tissue Beetles Tissue Beetles Tissue Beetles

L. terebrantis 18 3 45 62 0 0 G. huntii 3 2 13 43 0 0 L. procerum 1 0 10 12 9 0 L. serpens 0 0 0 2 0 0

1Sample trees lacking most (>90%) foliage, with the remaining being dead (red). 2Sample trees with predominantly (>70%) dying foliage (pale green and yellow). 3Sample trees with predominantly (>90%) healthy foliage (dark green).

Resumen Brown, H. D., and Mcdowell, W. E. 1968. Status of lob- lolly pine die-off on the Oakmulgee District, Tallade- Los escarabajos de la corteza en la familia Cur- ga National Forest, Alabama-1968. U.S. Dep. Agric. culionidae que se alimentan de raíces y los hongos For. Serv. Rept. No. 69-2-28. ophiostomatoides que ellos transmitan contribuyen Eckhardt, L. G., Goyer, R. A., Klepzig, K. D., and Jones, a los trastornos de las raíces de especies de Pinus J. P. 2004. Interactions of Hylastes species (Coleop- por todo el mundo. En el sudeste de los Estados tera: Scolytidae) with Leptographium species asso- ciated with loblolly pine decline. J. Econ. Entomol. Unidos, los escarabajos que alimentan de las raí- 97(2): 468-474. ces que pertenecen al género Hylastes (Coleoptera: Eckhardt, L. G., Weber, A. M., Menard, R. D., Jones, J. Curculionidae), incluyendo Hylastes salebrosus y P., and Hess, N. J. 2007. Insect-fungal complex asso- Hylastes tenuis, son asociados con los lotes de Pinus ciated with loblolly pine decline in central Alabama. taeda que estan experimentando la mortalidad pre- For. Sci. 53(1): 84-92. matura. A pesar de esto, evidencia directa de que Eckhardt, L. G., and Menard, R. D. 2008. Topographic Hylastes se este reproduciendo junto con los hongos features associated with loblolly pine decline in cen- ophiostomatoides en Pinus taeda no se ha observa- tral Alabama. For. Ecol. Mgt. 255: 1735-1739. do hasta este momento. En un lote de en Eckhardt, L., Sward Sayer, M. A., and Imm, D. 2010. P. taeda State of pine decline in the southeastern United Alabama que esta experimentando una mayor mor- States. Southern J. Appl. For. 34(3): 138-141. talidad, las raíces fueron excavadss y disectadas de Jacobs, K., and Wingfield, M. J. [eds.]. 2001. Lep- pinos que muestran una serie de condiciones de la tographium species, tree pathogens, insect associ- corona para determinar si la reproducción y el daño ates and agents of blue-stain. American Phytopath. causado por la alimentación de Hylastes se peude Soc. St. Paul, MN. ser detectado e investigado entre la relación de los Klepzig, K. D., Raffa, K. F., and Smalley, E. B. 1991: escarabajos, hongos ophiostomatoides y el estado Association of insect-fungal complex with red pine de la copa de de los árboles. Hylastes salebrosus y decline in Wisconsin. For. Sci. 37(4): 1119-1139. Klepzig, K. D., Smalley, E. B., and Raffa, K. F. 1995. H. tenuis representaba a la mayoría de los escara- Dendroctonus valens and Hylastes porculus (Co- bajos recolectados (94%) y fueron encontrados casi leoptera: Scolytidae): vectors of pathogenic fungi exclusivamente en las raíces de los árboles con coro- (Ophiostomatales) associated with red pine decline nas que estan muriendo. Los escarabajos adultos y disease. Great Lakes Entomol. 28(1): 81-87. las galerías de los escarabajos fueron asociados con Matusick, G., and Eckhardt, L. G. 2010. Variation in una gran cantidad de hongos de ophiostomatoides, virulence among four-root-inhabiting ophiostoma- incluyendo Grosmannia huntii, Leptographium toid fungi on Pinus taeda L., P. palustris Mill., and P. terebranis, L. procerum y L. serpens. Estas obser- elliottii Engelm. seedlings. Canadian J. Plant Path. vaciones sugieren que las raíces de los árboles que 32(3): 361-367. estan muriendo representan un substrato valioso Matusick, G., Somers, G. L., and Eckhardt, L. G. 2011. Root lesions in large loblolly pine (Pinus taeda L.) para la expansión de poblaciones de H. salebrosus y following inoculation with root-inhabiting ophiosto- H. tenuis. Un estudio más amplio es necesario para matoid fungi. For. Path. 42: 37-43. confirmar estos hallazgos. Menard, R. D. 2007. An assessment of the risk mapping Palabras Clave: galerías, Hylastes, disminu- system for the use of managing loblolly pine decline ción de pino loblolly, hongos ophiostomatoides, sites within red-cockaded woodpecker habitat. M.S. trastornos de raíces Thesis Louisiana State University. Baton Rouge, LA. Stoakley, J. T. 1968. Control of the pine weevil, Hy- lobius abietis L., and of Hylastes species. Forestry References Cited 41(2): 182-188. Thompson, J. A. 2011. Two-year bark and ambrosia bee- Blackman, M. W. 1941. Bark beetles of the genus Hylas- tle diversity study at the Talladega National Forest tes Erichson in North America. U.S. Dept. of Agric. in the Southeastern United States. M.S. Thesis, Au- Misc. Publ. no. 417. burn University, Auburn, AL. Scientific Notes 241

Witcosky, J. J., and Hansen, E. M. 1985. Root-colonizing optera: Curculionidae) as vectors of black-stain root insects recovered from Douglas-fir in various stages disease of Douglas-fir. Environ. Entomol. 15: 1090- of decline due to black-stain root disease. Phytopa- 1095. thology 75: 399-402. Zanzot, J. W., Matusick, G., and Eckhardt, L. G. 2010: Witcosky, J. J., Schowalter, T. D., and Hansen, E. M. Ecology of root-feeding beetles and their associated 1986. Hylastes nigrinus (Coleoptera: Scolytidae), fungi on longleaf pine in Georgia. Eviron. Entomol. Pissodes fasciatus, and (Cole- 39(2): 415-423.