Eugene B. Smalley

Interaction of pre-attack and induced monoterpene concentrations in host conifer defense against bark beetle-fungal complexes

Two pine species (Pinus resinosa, P. banksiana) responded to inoculation with fungi carried by bark beetles by rapidly increasing monoterpene concentrations at the entry site. Changes in total monoterpenes were more pronounced than changes in proportionate compositions. The extent and rate of host response was affected by fungal species, the viability of the inoculum, and host tree species. In general, host responses were highest to fungi that are phytopathogenic and consistently associated with the major bark beetles in the study region. Simple mechanical wounding cannot account for the observed allelochemical changes, as aseptic inoculations elicited only minor reactions. Similarly, inoculation with autoclaved inviable fungi generally elicited intermediate responses, suggesting that both structural and metabolic fungal properties are important. Responses by jack pine, P. banksiana, were generally more rapid and variable than those of red pine, P. resinosa. Dose-toxicity experiments with synthetic compounds demonstrated that monoterpene concentrations present in vivo only a few days after simulated attack are lethal to most beetles. Constitutive (pre-attack) monoterpene levels can also exert some toxicity. Because bark beetles engage in pheromone-mediated mass attacks that can deplete host defenses, constitutive monoterpene levels, while a necessary early phase of successful plant defense, appear insufficient by themselves. Such interactions between constitutive and induced defense chemistry may be important considerations when evaluating general theories of plant defense.

Effects of biotic and abiotic stress on induced accumulation of terpenes and phenolics in red pines inoculated with bark beetle-vectored fungus.

This study characterized the chemical response of healthy red pine to artificial inoculation with the bark beetle-vectored fungusLeptographium terebrantis. In addition, we sought to determine whether stress altered this induced response and to understand the implications of these interactions to the study of decline diseases. Twenty-five-year-old trees responded to mechanical wounding or inoculation withL. terebrantis by producing resinous reaction lesions in the phloem. Aseptically wounded and wound-inoculated phloem contained higher concentrations of phenolics than did constitutive tissue. Trees inoculated withL. terebrantis also contained higher concentrations of six monoterpenes,α-pinene,β-pinene, 3-carene, limonene, camphene, and myrcene, and higher total monoterpenes than did trees that were mechanically wounded or left unwounded. Concentrations of these monoterpenes increased with time after inoculation. Total phenolic concentrations in unwounded stem tissue did not differ between healthy and root-diseased trees. Likewise, constitutive monoterpene concentrations in stem phloem were similar between healthy and root-diseased trees. However, when stem phloem tissue was challenged with fungal inoculations, reaction tissue from root-diseased trees contained lower concentrations ofα-pinene, the predominant monoterpene in red pine, than did reaction tissue from healthy trees. Seedlings stressed by exposure to low light levels exhibited less extensive induced chemical changes when challenge inoculated withL. terebrantis than did seedlings growing under higher light. Stem phloem tissue in these seedlings contained lower concentrations ofα-pinene than did nonstressed seedlings also challenge inoculated withL. terebrantis. It is hypothesized that monoterpenes and phenolics play a role in the defensive response of red pine against insect-fungal attack, that stress may predispose red pine to attack by insect-fungal complexes, and that such interactions are involved in red pine decline disease. Implications to plant defense theory and interactions among multiple stress agents in forest decline are discussed.

Combined chemical defenses against an insect-fungal complex

This study considered how host plant allelochemicals may contribute to defense against insects and fungi that jointly colonize the subcortical tissues of trees, the relative roles of constitutive and inducible chemistry in these defenses, and how the actions of two different feeding guilds might be interrelated. Our model consisted of the coniferous treePinus resinosa, the root- and lower stem-colonizing beetlesHylastes porculus andDendroctonus valens, and their associated fungiLeptographium procerum andL. terebrantis, and the stem-colonizing bark beetleIps pini and its associated fungusOphiostoma ips. In a novel bioassay, extracts from reaction tissue elicted by wound inoculation withL. terebrantis were more repellent to beetles than were similar extracts from constitutive or mechanically wounded tissue. The effect on beetle behavior was more pronounced in nonpolar extracts, which contain mostly monoterpenes, than in polar extracts, which contain mostly phenolics. Synthetic monoterpenes at concentrations present in the various tissues exerted similar effects and were likewise repellent in dose-response experiments. Growth ofL. procerum andL. terebrantis was inhibited by polar extracts from constitutive and reaction tissue. Inhibition was higher in wounded than control tissue, but the inhibition response did not vary with the type of wounding. Synthetic monoterpenes strongly inhibited spore germination and mycelial growth of both fungi. Colonization of red pine roots byLeptographium spp. altered the subsequent effects of extracts of stem phloem tissue onI. pini. These effects varied with host condition. Beetles preferred extracts from constitutive stem phloem tissue of healthy trees to that of root-diseased trees. However, extracts from reaction tissues of healthy trees were more repellent toI. pini than were the reaction tissues of root-diseased trees. The implications of these results to plant defense against insect-fungal complexes and interactions among different feeding guilds are discussed.

Interactions of ecologically similar saprogenic fungi with healthy and abiotically stressed conifers

Abstract Saprogenic fungi are distinguished in part by their inability to cause disease in healthy hosts, and relatively high ability to kill or accelerate the decline of stressed hosts. We sought to determine the degree to which Leptographium terebrantis and L. procerum , ecologically similar, ophiostomatoid fungi, are saprogenic. In inoculation experiments, Leptographium terebrantis was better able to colonize roots of both mature and seedling Pinus resinosa than was L. Procerum . In addition, L. terebrantis colonized roots of shade-stressed seedlings to a significantly greater extent than it colonized roots of non-stressed seedlings. L. procerum exhibited no such trend. Due to its greater virulence within stressed than healthy hosts and related traits. L. terebrantis was judged to have a relatively high degree of competitive saprogenic ability as compared with L. procerum . This study indicates the biologically important differences which may occur between closely related, ecologically similar fungi and may have implications to studies of declines and diseases with which L. terebrantis and L. procerum have been associated.

Tibial Dyschondroplasia of Chickens Induced by Fusarochromanone, a Mycotoxin

Chicks were fed for 3 weeks a practical broiler starter diet amended with fusarochromanone (FC), a mycotoxin. In Hubbard broiler chicks, the highest test dose of FC (75 ppm) suppressed body weight by 33% and humoral response to sheep erythrocytes by 34% to 50%, and increased the incidence of tibial dyschondroplasia (TD) to 100%. The intermediate dose (35 ppm) significantly increased the incidence of TD in both types of broiler chicks (Hubbard and Indian River), but its effect on humoral response to sheep erythrocytes was not consistent across several experiments. A minimum dietary concentration greater than 20 ppm was required to significantly increase the incidence of TD. Excessive dietary copper or zinc (200 ppm) alleviated the effect of FC on TD. However, activities of copper-dependent ceruloplasmin and superoxide dismutase was not affected by FC. Cartilage mineral profiles in FC- or Thiram-induced TD differed from that of 1-day-old hypertrophic embryonic cartilage core (less K and Mn). FC did not induce TD in leghorn chicks.

Induction of Tibial Dyschondroplasia and Suppression of Cell-Mediated Immunity in Chickens by Fusarium oxysporum Grown on Sterile Corn

An isolate of Fusarium oxysporum from corn associated with Kaschin-Beck disease in humans was tested for its ability to induce tibial dyschondroplasia (TD) and toxicity in chicks. Both leghorn and broiler chicks were fed diets in which corn was replaced with varied amounts (0% to 50%) of the F. oxysporum culture grown on sterile corn, or with known TD-inducing agents. F. oxysporum did not affect body weight in either type of chicks. In leghorn chicks, neither F. oxysporum nor the known TD-inducing agents (F. equiseti, 4%; tetramethylthiuram disulfide [Thiram], 35 ppm) caused TD. However, F. oxysporum at high levels (50%) and the two known TD-inducing agents reduced interdigital cutaneous response to phytohemagglutinin-P challenge. In addition, Thiram also reduced body-weight gain by more than 17%. In female broiler chicks (Cornish Rock), F. oxysporum not only decreased cell-mediated cutaneous response to phytohemagglutinin-P but also increased TD incidence; these same effects were observed with F. equiseti and Thiram. Histological examinations revealed similar pathological changes among dyschondroplastic lesions induced by F. oxysporum, F. equiseti, and Thiram. Results of this experiment indicate that the isolate of F. oxysporum from the region in which Kaschin-Beck disease is endemic can induce TD in broiler chicks and that it is immunosuppressive.

Decreased Cell-Mediated Immunity and Lack of Skeletal Problems in Broiler Chickens Consuming Diets Amended with Fusaric Acid

Young female broiler chickens fed diets amended with 0, 35, 75, and 150 mg fusaric acid (FA)/kg diet for 3 weeks showed no aberrations in behavior, feed intake, weight gain, or appearance of the visceral organs. Furthermore, there was no correlation between the dietary concentration of FA and incidence of tibial dyschondroplasia and leg-shape deformities. Ash content of dry fat-free tibiae was not influenced by FA; thus, no rickets was present in these chickens. FA enhanced the humoral response to sheep erythrocytes but significantly reduced cell-mediated cutaneous response to phytohemagglutinin-P.

Prevention of Thiram-induced dyschondroplasia by copper

Abstract Broiler chicks were dietarily intoxicated with Thiram. The toxic effects observed included reduced ceruloplasmin activity and bone strength, and suppressed primary humoral immunity. In addition, Thiram induced a lesion characterized by abnormal cartilage formation in the proximal end of the tibiotarsus known as tibial dyschondroplasia. The incidence and severity of dyschondroplasia induced by Thiram was reduced by supplementing the diet with CuSO 4 , but not with ZnSO 4 or MnSO 4 . However, all three trace elements prevented the adverse effect of Thiram on primary humoral immunity. The prevention of toxicity of Thiram by supplementing the diets with trace elements strongly suggested that either Thiram interfered with the metabolism of trace elements or the trace elements rendered Thiram less toxic. Based on these observations, we conclude that the etiology of Thiram-induced dyschondroplasia is distinct from dyschondroplasia which is not prevented by dietary copper supplementation.

ELEVATED PLASMA GLYCOSAMINOGLYCANS IN CHICKENS WITH TIBIAL DYSCHONDROPLASIA INDUCED BY A FUSARIUM OXYSPORUM ISOLATE

Chicken plasma glycosaminoglycans (GAGs) were isolated and digested. Their building block molecules, namely, glucosamine and galactosamine, were quantified by gas chromatography. The levels of these two amino sugars were elevated in broiler chickens with tibial dyschondroplasia (TD) induced by culture material of Fusarium oxysporum (FO), a mold isolated from corn originated from the endemic region of Kaschin-Beck disease (KBD) in China. As the TD severity score changed from 1 (healthy) to 2, 3, and 4, glucosamine increased by 10%, 33%, and 57% and galactosamine by 9%, 13% and 48%, respectively. The elevated plasma GAGs correlated to TD severity but not to the amount of FO material in the diets. This correlation of plasma GAGs to TD in chickens parallels the reported correlation of urinary GAGs to KBD in humans. The possibility of TD as an animal model for KBD is discussed.