Progress 10/01/99 to 09/30/04
Outputs
What follows is a summary of this five-year project dealing with infestation of nursery trees in the Southeast by the Asian ambrosia beetle (Xylosandrus crassiusculus). Female ambrosia beetles migrate to tree trunks and branches in the spring and tunnel into the wood. Studies of other species of ambrosia beetles show the insects to be attracted to their host through ethanol released from the trees. We have observed that for certain trees (such as Kwanzan cherry) more than 80 percent of the insect bore holes are made at the site of lenticels. In red oak, 53 percent of infection sites were through lenticels, although lenticels accounted for only 2.5 percent of the surface area of the tree trunk. Since these structures serve for gas exchange between internal tissues of the trunk and the atmosphere, it is not difficult to imagine ethanol vapor being released through these same sites. Beetles carry with them fungi within a special sac or mycangium and also probably carry
fungi on their body surfaces. The resulting beetle infestation and fungal growth lead to wilt symptoms in the host. Microscopy of infected wood shows a mixture of bacteria, yeast, and filamentous fungi with the latter predominating. Fungal hyphae pass from one host cell to another either through pit membranes, through perforations of vessel members, or directly through cell walls. The cytoplasm of wood parenchyma cells probably provides the food source for continued hyphal growth. Parenchyma cell walls degrade into a gel which is secreted and blocks water-conducting vessels in the wood. However, this response can be elicited in wood by drilling sterile holes to mimic beetle tunnels. Thus, vessel blockage probably is induced both by formation of the tunnels as well as by the activities of the fungi. During the first two years of identifying the fungal pathogens, Ambrosiella xylebori was consistently associated both with captured beetles and with infected wood. This fungal species is
specifically associated with the Asian ambrosia beetle in Japan. These beetles were first reported in the U.S. in 1974, but wood containing A. xylebori was first collected in 1967; therefore, the beetle probably existed in the U.S. for a few years before its discovery. Surface sterilization studies indicate that this fungus is not carried on the surface of the beetle but probably in the mycangium. Unfortunately, during the last two years we have been unable to isolate A. xylebori either from beetles or wood. The reasons for this are unknown. Instead, accessory fungi were isolated including: Fusarium oxysporum, and species of Pestalotia and Phomopsis. Saplings of redbud were inoculated with these fungi by drilling artificial tunnels and inserting a toothpick smeared with the fungus. In all instances, later anatomical investigation of the wood or fungal re-isolation in culture indicated that colonization had been successful although the saplings had remained asymptomatic. Either these
accessory fungi do not cause the disease in nature or perhaps the beetle vector provides a substance to encourage fungal growth and/or disease symptoms in the host.
Impacts
A knowledge of which fungus is responsible for the wilt disease should allow more selective and effective treatment of infested trees.
Publications
- Dute, R. R., M. E. Miller, M. A. Davis, F. M. Woods and K. S. McLean. 2002. Effects of ambrosia beetle attack on Cercis canadensis. International Association of Wood Anatomists Journal 23: 143-160.
- Dute, R. R., M. E. Miller, M. A. Davis, F. M. Woods and K. S. McLean. 2002. Fungal growth and host response associated with ambrosia beetle attack on Cercis canadensis. Journal of the Alabama Academy of Science 73(2-3), 67 (abstract).
- Dute, R. R., B. L. Prather and M. J. Valente. 2004. An update on fungal infections associated with ambrosia beetle infestations. Journal of the Alabama Academy of Science 75(2), 67 (abstract).
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Progress 01/01/03 to 12/31/03
Outputs
Xylosandrus crassiusculus, the Asian ambrosia beetle, is a widespread pest in tree farms in the Southeast. This beetle carries with it the fungus Ambrosiella xylebori which infects the wood as the beetle tunnels into the host. The fungus serves as a food source for the beetle, and the tree shows symptoms of wilt. There is controversy as to whether the wilt is caused by Ambrosiella or by secondary fungi, such as Fusarium, which are introduced by the beetle or which enter the tunnel at a later time. This spring we were unable for the first time to isolate Ambrosiella either from beetles in flight or from infested trees (overcup oak) showing signs of wilt. In the latter instance, Fusarium oxysporum was a common isolate, and we decided to attempt re-infection of a woody host. Eight, container-bound saplings of Cercis canadensis (redbud) were used. Two saplings remained untreated, two received drill holes without inoculum (to simulate beetle damage), and four received
drill holes with inoculum. Two-millimeter diameter holes were drilled (one hole per internode) in a spiral pattern up the trunk for a total of five holes per bole. Inoculum was from a PDA-grown culture of F. oxysporum isolated from the aforementioned overcup oaks. All holes were covered with parafilm. After seven weeks the saplings were felled and a disk 2 cm thick was cut at 2 cm above each drill hole. Untreated controls had disks cut at comparable heights. In the laboratory each disk was cut into two semicircles along the line of the drill hole. One half of each disk was processed for fungal growth and culturing as described in a previous report. The other half was preserved in formalin-acetic acid-alcohol (FAA). These latter specimens were later softened in water and sectioned at 20-40 micrometers transversely with a sliding microtome. The wood sections were then dehydrated, mounted on slides, and viewed unstained for the presence of hyphae. Although the saplings were asymptomatic
at the time of collection, the culture experiments showed the presence of F. oxysporum only in the inoculated specimens. Anatomical studies of the sectioned material showed inoculated woods whose vessels contained hyphae as well as a brown, gummy substance that (in a previous study) was observed to be secreted by parenchyma cells surrounding the vessels. However, a similar secretion was also found in those specimens that had received drill holes without inoculum. Thus, the secreted material represents a general response to wounding and not a specific response to F. oxysporum. It is unknown why colonization of the wood occurred but the trees remained asymptomatic. We plan to repeat the re-infection experiment in the coming year using F. oxysporum from infected trees and also using A. xylebori if it is available.
Impacts
A knowledge of which fungus is responsible for the wilt disease should allow more selective and effective treatment of infested trees.
Publications
- No publications reported in 2003.
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Progress 01/01/02 to 12/31/02
Outputs
In an effort to pinpoint the wilt-causing fungus associated with ambrosia beetle infestations of nursery trees, we repeated a previous laboratory experiment. Four wafers of wood from each of five infested (wafers collected 1 cm from beetle tunnels) and three uninfested (control) redbud trees were collected. Each wafer was subdivided into 10 to 20 one-half centimeter square tissue sections. Sections were surface sterilized and placed onto potato dextrose agar for 5 to 7 days at 24 C. During this time any fungi growing from the wood sections were identified. Only fungi of the genus Ambrosiella were consistently associated with beetle infested wood and not with controls. In contrast to our previous supposition, only one species of Ambrosiella, A. xylebori, is present in infested wood. In Japan, this fungus is known to associate specifically with the beetle Xylosandrus crassiusculus, the same beetle involved in the infestation here in the Southeast. However, the first
report of beetle infestation was noted in South Carolina in 1974, whereas the type specimen for the fungus was collected in 1967 in Georgia. Therefore, the beetle must have been in the southeastern U.S. for some years before its discovery. A number of beetles were trapped in the spring before they could attack trees. To detail the association between beetle and fungus 1) 60 non-sterile beetles, 2) 110 intact, surface-sterilized beetles, and 3) 35 surface-sterilized, dissected beetles were plated onto potato dextrose agar and the resulting fungal colonies identified. In 1), 3 beetles produced Ambrosiella colonies, but all specimens produced other fungi as well. In 2), 28 of the beetles produced Ambrosiella colonies exclusively, 1 produced only Fusarium, and 10 produced only Penicillium. In 3), 3 beetles produced only Ambrosiella colonies. From these results, it would seem that Ambrosiella is carried within the beetles (probably within the mycangia), whereas most other fungi that enter
the tree as beetles excavate their tunnels are probably localized on the surface of the insects. We next plan to inoculate trees with colonies of Ambrosiella in an effort to reproduce disease symptoms.
Impacts
A knowledge of the disease fungus plus how, when, and where it is carried should aid in designing control measures.
Publications
- Dute, R. R., M. E. Miller, M. A. Davis, F. M. Woods and K. S. McLean. 2002. Effects of ambrosia beetle attack on Cercis canadensis. International Association of Wood Anatomists Journal 23: 143-160.
- Dute, R. R., M. E. Miller, M. A. Davis, F. M. Woods and K. S. McLean. 2002. Fungal growth and host response associated with ambrosia beetle attack on Cercis canadensis. Journal of the Alabama Academy of Science 73 (2--3) (abstract).
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Progress 01/01/01 to 12/31/01
Outputs
As part of a continuing investigation of Asian ambrosia beetle infestation of nursery trees in the Southeast, infected wood was collected and returned to the laboratory in an attempt to culture and identify fungi associated with beetle tunnels. Trunk portions from separate infected individuals of redbud (4), Kwanzan cherry (2), and crepe myrtle (2) were collected. At the same time a trunk portion from an uninfected tree was collected for each species. In the laboratory, a thin cross-sectional wafer of wood (1-2 mm thick) was removed from each infected trunk at a distance 1 cm above or below an ambrosia beetle tunnel. It was felt that this procedure would select for disease fungi that had gone systemic. Each wafer was subdivided into 10 to 25 one-half centimeter square tissue sections. After surface sterilization, tissue sections were placed on potato dextrose agar. Cultures were incubated for 5 to 7 days at 24 C during which time any fungi growing from the wood
specimens were identified or subcultured for later identification. Species of Ambrosiella were associated with the infected trunk portions of all tree species investigated. Two unidentified species of Ambrosiella (A and B) were present. All infected trunks had either species A, species B, or both. No colonies of this fungus were isolated from the controls. Species of Fusarium, another fungal genus often associated with beetle infection, were not found in cherry, were found in two of four trunks of redbud, and were found (in considerable quantity) in both trunks of crepe myrtle. However, in the latter instance, the control also contained Fusarium. A number of other fungal genera were isolated from both infected and control woods but were not thought to be specifically associated with the wilt symptoms of the host. The beetle infestation in this experiment was well-established and probably secondary infections appeared as the tunnels aged. We plan to repeat this experiment on newly
formed tunnels and to culture beetles captured before infesting a tree.
Impacts
A knowledge of the fungi involved should provide insight into the basic disease mechanism and might aid in proposing control measures.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
Damage caused to Cercis canadensis (redbud) by the Asian ambrosia beetle and its associated micro-organisms was investigated as was the host response to infestation. Various types of micro-organisms were introduced by beetles but only hyphal fungi exhibited extensive growth. The fungi, of ascomycetous ultrastructure, infected all cell types by penetrating pit membranes. Infestation of parenchyma cells was especially intensive and these cells probably provided the food source for continued hyphal growth. Parenchyma cells responded to damage by degradation and gelation of their protective layers. The resulting carbohydrate slime filled the parenchyma cells and passed into and occluded associated vessels. Measurements showed no significant difference in ethylene production by wood samples from infected versus uninfected trees. However, the established literature would indicate that damage-induced ethylene production was responsible for initiating events that resulted in
vascular blockage by carbohydrate gel.
Impacts
Although episodic in nature, during years of heavy beetle infestation economic losses to nurseries in the Southeast can be severe. This last year we have gained an understanding of the response of the woody host to infestation. In the coming year we plan to focus on the fungi responsible for the infection.
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
Because this project started in October 1999, all progress will be reported in the 2000 AD-421 report.
Impacts
(N/A)
Publications
- No publications reported this period
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