Oxalic Acid as a Fungal Acaracidal Virulence Factor

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1 VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS Oxalic Acid as a Fungal Acaracidal Virulence Factor BRETT H. KIRKLAND, ASMA EISA, AND NEMAT O. KEYHANI 1 Department of Microbiology and Cell Science, University of Florida, Gainesville, FL J. Med. Entomol. 42(3): 346Ð351 (2005) ABSTRACT Cell-free culture supernatants of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin were able to induce mortality in several tick species, including adult Ambylomma americanum (L.), Ambylomma maculatum Koch, and Ixodes scapularis Say. Four lines of experimental evidence support the hypothesis that oxalic acid secretion by B. bassiana, coupled to a reduction in the ph of the medium, act as potent acaricidal factors during pathogenesis. 1) Acaracidal activity of culture supernatants was retained after treatments including boiling and protease digestion, but was lost after dialysis. 2) Metabolite analyses indicated oxalate to be the major secreted organic compound present in the active culture supernatants. 3) Treatment of ticks with the pure compound oxalate at ph 4.0 resulted in almost 80% mortality in adult A. americanum ticks within 14 d, whereas treatment of ticks with oxalate at ph 7.0, or with formate, citrate, or phosphate at both ph 4 and 7 resulted in 10% mortality, even after 28 d. 4) Cell-free culture supernatants from B. bassiana mutants with decreased oxalate production displayed lower acaricide activity than wild type. KEY WORDS tick biocontrol, entomopathogenic fungi, Beauveria bassiana, oxalate, ph ENTOMOPATHOGENIC FUNGI SUCH AS Beauveria bassiana (Bals.) Vuillemin and Metarhizium anisopliae Metschnikoff (Ascomycota: Hypocreales) are pathogenic toward several hard ticks of the family Ixodidae, including Ixodes scapularis Say, Boophilus decoloratus Koch, Rhipicephalus appendiculatus Neumann, Amblyomma variegatum F., and Amblyomma maculatum Koch (Chandler et al. 2000, Kaaya 2000, Samish 2000, Benjamin et al. 2002, Fernandes et al. 2003). Ticks represent important vectors of animal and human infectious disease agents, and effective reduction and control of tick populations remain difþcult (Sonenshine 1993, George 2000, Pegram et al. 2000, Eisler et al. 2003). Although the use of entomopathogenic fungi as additional tools for integrated pest management practices holds promise, several tick species, including Dermacentor variabilis (Say) and Amblyomma americanum (L.), seem to have a level of resistance to fungal infection. Intriguingly, this resistance can be overcome by speciþc inoculation conditions (Kirkland et al. 2004a, b). Little to no mortality was observed using B. bassiana fungal conidia or blastospores washed into distilled H 2 O (dh 2 O) buffer, or with fresh media, but 50% mortality within 14Ð28 d of treatment was observed when ticks were treated with fungal cells with growth media carryover. These data indicated that B. bassiana secretes important virulence factors that can enhance or enable pathogenesis or that successful infection requires some exogenous nutrient source (Kirkland et al. 2004a, b). In this regard, B. bassiana is known to secrete a suite of proteases, glycosidases, 1 Corresponding author, keyhani@uß.edu. and lipases as well as toxic metabolites during the infection process (Kucera and Samsinakova 1968, Roberts 1981, St Leger et al. 1986, Charnley and St Leger 1991, Gupta et al. 1992, Clarkson and Charnley 1996, St Leger et al. 1997). Oxalate is produced in both plants and fungi, and it is one of the major organic acids secreted by a variety of fungal species (Kubicek 1987, Gadd 1999). Its production in fungi is often related to metal detoxiþcation and pathogenesis, with the latter due to effects that include acidiþcation of host tissues; sequestration of metal ions such as calcium, manganese, magnesium, and iron; and possible inhibition or disruption of host defense responses (Godoy et al. 1990, Gadd 1999, Cessna et al. 2000, Jarosz-Wilkolazka and Gadd 2003). As a phytopathogenic fungal virulence factor, oxalic acid seems to facilitate disruption of the plant cell wall while promoting the functions of fungal lytic enzymes that ultimately lead to wilting and disease progression (Bateman and Beer 1965, Magro et al. 1988, Munir et al. 2001). Conversely, high concentrations of oxalate in plants such as rhubarb, spinach, and beets are thought to discourage insect foraging, although the exact physiological roles of plant oxalates on insects is unknown (Oke 1969, Franceschi and Horner 1980, McConn and Nakata 2002). Oxalate crystals have been identiþed on cadavers of insects infected by fungi, including B. bassiana (Moino et al. 2002). Oxalic acid is toxic toward the honey bee mite Varroa destructor Anderson & Trueman and has been demonstrated to reduce the biological Þtness of the tarnished plant bug, Lygus hesperus (Palisot de Beauvois) (Alverson 2003, Gregorc and Poklukar /05/0346Ð0351$04.00/ Entomological Society of America

2 May 2005 KIRKLAND ET AL.: OXALIC ACID AS A FUNGAL ACARICIDE ). Metabolic acids, including oxalate, have been implicated in B. bassiana-mediated virulence toward the migratory grasshopper, Melanoplus sanguinipes (F.) (Bidochka and Khachatourians 1991). In this study, we report that oxalic acid displays a ph-dependent toxicity toward ticks (adult A. americanum) and that secretion of oxalic acid by B. bassiana may help account for the acaracidal activity of cell-free fungal culture supernatants. These data support the hypothesis that oxalate is an important entomopathogenic fungal virulence factor, similar to observations concerning the importance of oxalate during phytopathogenic fungal infection of plant hosts. Materials and Methods Oxalate ( 99% purity) and other chemicals and reagents were purchased from Sigma (St. Louis, MO) and Fisher (Pittsburgh, PA). Unfed adult A. americanum, A. maculatum, and I. scapularis were obtained from Oklahoma State University Tick Rearing Facility (Stillwater, OK). For inoculation into the various liquid media, B. bassiana (ATCC 90517) was grown on potato dextrose agar (PDA) containing 5 g/ml (Þnal) trimethoprim. Plates were incubated at 26 C for 10Ð12 d, and conidia were harvested by ßooding the plate with sterile dh 2 O. Conidial suspensions were Þltered through glass wool, and Þnal spore concentrations were determined by direct count by using a hemocytometer. Liquid broth cultures were inoculated (1:20) with conidia harvested from plates to a Þnal concentration of 0.5Ð conidia/ml. Cultures were grown at 26 C with aeration (175Ð200 rpm, Innova 4230 culture shaker, New Brunswick Science, Edison, NJ). Fungal cell mass was removed from cultures by centrifugation (10,000 g, 15 min), and the cell culture supernatant (spent media) was Þltered through a 0.22-mm sterilization membrane. All treatments were performed using freshly prepared spent media except where indicated. The effect of storage temperature was tested on aliquots of the spent culture supernatant stored at either 4 Cor 20 C. Spent media were isolated from cultures grown in Sabouraud dextrose (SD), Sabouraud dextrose 0.5% yeast extract (SDY), Czapek-Dox (CzD), and potato dextrose (PD) broths. Samples used for tick treatments were isolated from 6-d cultures, and the supernatants of the wild-type and mutant strains (described below) were analyzed over a time course including 3, 5, 6, 7, 9, 10, and 14 d of growth in media. Where indicated, culture supernatants (6 d) were treated with proteinase K (MP Biomedicals, Aurora, OH) as follows: samples (2 ml) were incubated with 100 l of 10 mg/ml proteinase K solution (dissolved in dh 2 O)for1hat37 C. Ticks were submerged for 60 s in experimental solutions, and excess liquid was removed with either a pipet or a cotton swab. Test solutions included, B. bassiana culture supernatants, 50 mm sodium oxalate adjusted to ph values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 by using either NaOH or HCl, and 1, 5, 10, 20, and 50 mm solutions of sodium oxalate, sodium formate, sodium phosphate, and sodium citrate adjusted to ph values of 7.0 and ph 4.0 with either NaOH or HCl. Each experiment included 20Ð50 ticks, and experiments were repeated three times. Ticks were placed in conical tubes or microtiter plates containing numerous needle puncture holes (to allow for free ßow of air exchange) and stoppered with Styrofoam plugs. Specimens were placed in a humidity chamber ( 90% RH) with a 12-h day (27 C)/night (25 C) cycle, with mortality recorded every day. Aliquots of cell-free culture supernatants were analyzed for carbohydrates and organic acids by highperformance liquid chromatography (HPLC, HP Series II 1090, Hewlett Packard/Agilen, Wilmington, DE) by using an Aminex HPX-87H column (Bio-Rad) run isocratically in 4 mm H 2 SO 4 and coupled to both UV and refractive index detectors. The oxalate peak was quantiþed using a standard curve generated using the chemical compound. An aliquot of each supernatant was Þltered through a 5,000-molecular weight cut-off membrane (VivaScience, Binbrook Hill, Lincoln, United Kingdom), and the Þltrate was acidiþed by addition of dilute sulfuric acid before injection (10Ð20 l) onto the column. Chemical mutants of B. bassiana strain were produced using the alkylating reagent ethyl methanesulfonate (EMS) essentially as described by St Leger et al. (1999). Brießy, a spore suspension (0.1 ml of 1Ð conidia/ml) was added to 0.9 ml of 50 mm potassium phosphate buffer, ph 7.0, containing 10 l of EMS. Cells were incubated at 26 C for 5Ð8 h with aeration. Samples (0.1Ð0.5 ml) were diluted 1:10 into buffer containing 10% (wt:vol) sodium thiosulfate and incubated for an additional 0.5Ð1 h with aeration. Samples were diluted (typically to 0.5Ð viable cells/ml) and plated onto selection media (SDY containing 0.01% bromocresol purple; adjusted to ph 6.8). Plates were incubated at 26 C for 8Ð10 d. Under these conditions, the wild-type B. bassiana strain produced yellow halos within 3Ð5 d. Colonies that lacked or had reduced zones of yellow (on ph 6.8 plates) were removed, and single spores were isolated and rescreened on solid ph-indicator media over several generations (three to Þve times). Results Our previous results had indicated that addition of spent culture supernatant could increase fungal mediated mortality toward certain tick species (Kirkland et al. 2004a, b). To our surprise, adult A. americanum were susceptible to cell-free culture supernatants derived from growth of the entomopathogenic fungi B. bassiana in different media (Table 1). High mortality was observed within 14 d by using fungal spent media isolated from 6-d cultures grown in SD or SDY media, with lower mortality seen using spent PD media, and little to no mortality observed using culture supernatants from CzD media. A second treatment with media with SD or SDY spent media applied 14 d after the initial treatment resulted in up to 65Ð85% (total) mortality within 28 d of the original treatment, whereas

3 348 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 42, no. 3 Table 1. Acaracide activity towards adult A. amblyomma, oxalate concentration, and ph of cell-free B. bassiana culture supernatants nd treatment (on day 21) Spent growth media a (6 d culture supernatants) % mortality b Oxalate c ph d A. americanum, 14 d mm spent culture SD SD 1% yeast extract PD CzD a Fungal cells were removed from the liquid cultures by centrifugation. The resultant supernatants were Þltered through 0.22-mm Þlters and stored until use. b In all instances, 5% tick mortality was observed in control experiments using fresh media or sterile dh 2 O over the time course of the experiment. c No oxalate was detected in fresh media. d Initial ph values for the media were 5.6 (SD, SD YE, and PD) and 7.3 (CzD). second applications of spent PD or CzD media did not result in any increased mortality. Similar experiments treating adult A. maculatum or I. scapularis ticks with SDY supernatants resulted in and 32 15% (14 d posttreatment) mortality, respectively. Control treatments with sterile media or dh 2 O resulted in 5% mortality throughout the time course of the experiments. To test the heat lability of the observed acaricide activity, aliquots of SD or SDY 6-d culture supernatants were boiled for 10 min, allowed to cool to room temperature for 10 min, brießy spun to remove any precipitation, and then used to treat adult A. americanum ticks. Boiled supernatants resulted in 18 5 and 40 10% mortality after 14 d, respectively. To test whether the acaricide activity was primarily proteinaceous in nature, aliquots of SD and SDY culture supernatants treated with proteinase K (1 mg/ml) for 1hat37 C resulted in 24 6 and 50 10% mortality (14 d posttreatment). By contrast, dialysis using a 10,000-molecular weight cut-off membrane of active culture supernatants against 50 mm Tris buffer, ph 7.0 (10-ml aliquot versus 2 by 2 liters, overnight), resulted in marked reduction in the acaricide potency of the culture supernatants, with total mortality percentages dropping to 5 3% for SD and 9 3% for SDY media, 14 d posttreatment of adult A. americanum ticks. These data pointed toward the likely existence of small heatstable acaricide compound(s) secreted by B. bassiana into SD and SDY media during growth. Acaricide activity was stable to repeated freeze thawing cycles (at least three) and at 4 C for a least 1 mo. Metabolite analysis of culture supernatants by HPLC revealed oxalate to be the major organic acid present in the spent media. The oxalate concentrations as well as the ph of the culture supernatants used to treat the ticks were determined (Table 1). The concentration of oxalic acid and the resultant decrease in ph correlated with the acaricide activity of the culture supernatants. High concentrations of oxalate (20Ð35 mm) were produced when the fungal cells were grown in SD or SDY media, whereas lower %Mortality Time (d) Fig. 1. Oxalic acid-induced mortality in adult A. americanum ticks. Ticks were treated with solutions of 50 mm oxalate, ph 4.0 ( ); 20 mm oxalate ( ), ph 4.0; and 50 mm oxalate, ph 7.0 (Œ); all other conditions tested including 1, 5, 10, 20, and 50 mm citrate, ph 4.0 and 7.0, 1, 5, 10, 20, and 50 mm formate, ph 4.0 and 7.0, 1, 5 and 10 mm oxalate, ph 4.0, and 1, 5, 10, and 20 mm oxalate, ph 7.0 (dashed lines between the X marks). Values given are means of three experiments SE. amounts of oxalate ( 10 mm) were secreted when the cells were grown in PD, and almost no oxalate was produced when the cells were grown in CzD ( 0.5 mm) under the conditions tested. The acaracidal toxicity of oxalate was tested by using the chemical compound to treat adult A. americanum ticks (Fig. 1). Greater than 60% (14 d posttreatment) and 20% mortality was observed using 50 and 20 mm oxalate, ph 4.0, respectively. No signiþcant mortality was observed using a single treatment with lower concentrations of oxalate (1Ð10 mm), ph 4.0, or with solutions of oxalate at ph 7.0 (1Ð50 mm). Furthermore, no acaracidal activity was observed using single treatments of solutions of either of citrate, formate, or phosphate at ph values of either 4.0 or 7.0 and using concentrations up to 50 mm (Fig. 1). A second treatment 21 d after the initial treatment resulted in 75Ð85% total mortality by using either 20 or 50 mm oxalate, ph 4.0, and 40% mortality by using 50 mm oxalate, ph 7.0, within 14 d (35 d total). The ph dependence of the acaracidal activity of oxalate was investigated using oxalic acid solutions ranging from ph 4.0 to 7.0 (Fig. 2). Acaracidal activity was highest at ph 4.0 ( 80% mortality, 14 d posttreatment) with tick mortality rapidly decreasing as the ph of the oxalate solution was raised. Mortality at ph 4.5 was four-fold lower than that observed for solutions of oxalate at ph 4.0. Second treatments, 14 d after the Þrst, had a minor effect, resulting in up to 40% total mortality. To assess whether oxalate production by B. bassiana was indeed a contributing factor in the acaracidal activity of culture supernatants, mutant screens were established to isolate oxalate nonproducers. Because oxalate is one of the secreted organic acids responsible for the acidiþcation of the media, surviving colonies of EMS-treated conidia were plated onto SDY media supplemented with the ph indicator dye bromocresol purple (ph initial 6.8). Approximately 5,000 mutant

4 May 2005 KIRKLAND ET AL.: OXALIC ACID AS A FUNGAL ACARICIDE 349 clones of B. bassiana strain were screened on the indicator plates, and six mutants were identiþed that lacked or had reduced zones of surrounding yellow (acidiþcation). Of these, three seemed to be false positives and produced wild-type yellow zones when single spores were isolated and rescreened. The remaining three mutants, designated as clones A1 15, A1 16, and A1 17, seemed to retain their phenotype (no yellow zone of clearing, purple colonies) after at least three generations of rescreening on the indicator plates. Mutants A 15 and A1 16 displayed altered conidiation effects, forming smaller colonies that grew slower but sporulated more rapidly than wild type on PDA, SDA, and SDYA plates. Mutant A1 17 also displayed altered colony morphology but sporulated poorly under any of the conditions tested. The concentration of oxalate secreted by the mutants was quantiþed over a 15-d time course of growth in SDY broth (Fig. 3). These data indicated that two of the isolates, A1 15 and A1 16 produced no detectable oxalate under the conditions tested. Interestingly, the third mutant (A1 17) was able to produce oxalate at approximately one-half the levels as that of the wild-type strain. Culture supernatants (day 6) from the three mutants were used to treat adult A. americanum ticks in mortality experiments as described previously. Less than 10% mortality toward A. americanum ticks was observed using culture supernatants derived from any of the mutants grown in SDY including, A1 15, A1 16, and A1 17, even though the oxalate concentration in the latter supernatant approached 12 mm. Fig. 2. ph dependence of oxalate-induced mortality in adult A. americanum ticks. Mortality of adult A. americanum 14 d after treatment with 50 mm solutions of oxalate at the indicated ph values ( ), and mortality 14 d later, after a second treatment on day 14 (28-d total, f). Values given are means of at least three experiments SE. Fig. 3. Concentration of oxalate secreted into the medium during growth in SDY broth, wild-type B. bassiana (F), mutants A 15 and A 16 (E), and mutant A 17 (f). Discussion Although oxalic acid has been demonstrated to be an important virulence factor for the successful pathogenesis of phytopathogenic fungi during plant host infection, many plant species themselves produce oxalic acid, presumably to discourage insect foraging. Studies using oxalic acid have demonstrated oxalic acid to be toxic toward several insect species, including the tarnished plant bug (Alverson 2003) and the migratory grasshopper (Bidochka and Khachatourians 1991). In the latter report, metabolic acids produced by B. bassiana (including oxalate and citrate) acted synergistically with fungal conidia to promote successful pathogenesis. With respect to the Acari, oxalic acid has been used to control varroosis, and practical applications have demonstrated reduced infestations of V. destructor in bee colonies under conditions that present low toxicity toward the bees themselves (Gregorc and Poklukar 2003). Interestingly, entomopathogenic fungi such as M. anisopliae and B. bassiana have demonstrated virulence toward Varroa species, and the fungus also has been used to control these mites in bee colonies (Kanga et al. 2002, 2003). A. americanum ticks were predominately used in our studies due to the observation that this species can resist fungal (B. bassiana and M. anisopliae) infection (Kirkland et al. 2004b). Our results indicate that this resistance can be overcome and that (cell-free) culture supernatants derived from the entomopathogenic fungus B. bassiana can be toxic toward these ticks (and others), depending upon the fungal growth and media composition. HPLC analysis of culture supernatants coupled to tick mortality experiments conþrmed that one of the major acaricidal active ingredients in these supernatants was oxalate, although during fungal infection, secretion of factors such as hydrolytic enzymes, including proteases, glycosidases, and lipases, as well as other biologically active small molecules and toxins undoubtedly contributed to the establishment and progression of disease. Despite the relatively simple chemical formula (COOH) 2, oxalate is unique in that it displays at least three important chemical properties: it can act as a proton donor, an electron donor, and as a strong chelator of divalent cations. Our results indicated that oxalate toxicity was ph dependent, with mortality rates dramatically decreasing at ph 4.5 (single application). These data suggest that (as a diprotic compound with pk a values of 4.2 and 1.29) the reducing potential of oxalate may be an important factor in its tick toxicity. The relatively high concentration of oxalate, 50 mm, required for inducing mortality (in single treatments) may suggest that for the fungal organism, oxalate acts synergistically with other factors

5 350 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 42, no. 3 in promoting pathogenesis. Oxalate concentrations in culture supernatants did approach 30Ð35 mm, and it is possible that local oxalate concentrations during the infection process could be appreciably higher. Furthermore, hosts are likely to be continuously exposed to the secreted metabolites (including oxalate) that is likely to increase their toxicity. Indeed, oxalic acid is able to solubilize several components of insect cuticles, including elastin and collagen, and has been demonstrated to disrupt the integrity of M. sanguinipes cuticle directly (Bidochka and Khachatourians 1991). Using SDY ph indicator plates, three B. bassiana EMS-derived mutants were isolated displaying lowered levels of secreted oxalate (two of which produced 1% of the wild-type levels of oxalate under the conditions tested). Culture supernatants derived from all three mutants were nontoxic toward A. americanum ticks. Although these observations support the hypothesis that oxalate is an important fungal virulence factor during pathogenesis toward ticks, some caution should be taken in interpretation. Primarily, oxalate may act as a marker for other fungal factors required for pathogenesis, and disruption of ph pathways may have pleiotropic effects. In M. anisopliae, oxalate production and the resultant reduction in extracellular ph are linked to protease production and activity (St Leger et al. 1999), mutants unable to acidify the media also were deþcient in protease activity. This is similar to observations concerning phytopathogenic fungi where the secretion of oxalic acid leads to an acidic environment required for the expression and activities of many hydrolytic enzymes (Bateman and Beer 1965, Rollins and Dickman 2001). The virulence of the B. bassiana mutants was not assessed directly (i.e., by application of fungal cells to ticks) because these clones probably contain multiple mutations that would have obscured interpretation of any results, particularly because the mutants displayed altered developmental and conidiation phenotypes. Future research using targeted gene knockouts of enzymes in the oxalate biosynthetic pathway(s) (see below) of B. bassiana will probably help in understanding the physiological role of oxalate during pathogenesis. Several pathways exist in fungi for oxalate biosynthesis. In Aspergillus niger, oxaloacetate hydrolase can catalyze the conversion of oxaloacetate to oxalate and acetate (Kubicek 1987), whereas species of the phytopathogenic fungus Sclerotium can oxidize glyoxylate via the activity of a glyoxylate dehydrogenase (Maxwell and Bateman 1968, Balmforth and Thomson 1984). These systems link oxalate production to the tricarboxylic acid (TCA) and glyoxylate cycles, respectively. However, A. niger also possesses both a cytoplasmic pyruvate decarboxylase and oxaloacetate acetylhydrolase that would be capable of forming oxalate without the reactions of the TCA cycle (Kubicek et al. 1988). Finally, in wood-rotting fungi such as Fomitopsis palustris Gilbn. and Ryv., at least two additional oxalate-yielding routes, glyoxylate oxidase/ oxaloacetase) and a ßavohemoprotein glyoxylate dehydrogenase, have been described (Munir et al. 2001). Although the oxalate biosynthetic pathway in B. bassiana remains to be elucidated, preliminary mapping experiments have indicated the putative presence of at least the cytoplasmic pathway similar to that described above for A. niger (Cho and N.O.K., unpublished data). Finally, our results indicate that examining inoculum conditions that would favor oxalate production could increase the efþcacy of Þeld applications of B. bassiana in biocontrol efforts. This increase may be achieved by the selection of oxalate-producing constitutive strains, optimization of the conditions for oxalate production in already used strains, or even manipulation of dispersion formulas that maximize rapid oxalate production. Acknowledgments We thank J. Rollins for comments during manuscript preparation. This work was supported by the University of Florida and National Science Foundation. This paper is Florida Agricultural Experimental Station Journal series number R References Cited Alverson, J Effects of mycotoxins, kojic acid and oxalic acid, on biological Þtness of Lygus hesperus (Heteroptera: Miridae). J. Invertebr. Pathol. 83: 60Ð62. Balmforth, A. J., and A. Thomson Isolation and characterization of glyoxylate dehydrogenase from the fungus Sclerotium rolfsii. Biochem. 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