Journal of Undergraduate Research
Volume 7, Issue 2 - January/February 2006

Analysis of the Role of Oxalic Acid in the Entomopathogenic Fungus Beauveria bassiana

Asma N. Eisa

ABSTRACT

The role of oxalic acid in the pathogenicity of the fungus Beauveria bassiana towards ticks, including Ambylomma americanum, Ambylomma maculatum and Ixodes scapularis, was investigated. Cell-free culture supernatants of B bassiana were able to induce mortality in a number of tick species. Through experimental evidence, oxalic acid has been identified as a major acid waste product of B bassiana, its production depends on growth and nutrient conditions. Treatment of ticks with the pure oxalate, at pH 4.0 resulted in almost 80% mortality in adult A. americanum ticks within 14 days, whereas treatment of ticks with oxalate at pH 7.0, or with formate, citrate, or phosphate at pH values of both 4 and 7 resulted in less than 10% mortality even after 28 days. Cell-free culture supernatants from B. bassiana mutants with decreased oxalate production displayed lower acaracidal activity than wild-type. These results support the hypothesis that oxalic acid secretion by B. bassiana, coupled with a reduction in the pH of the medium, act as potent acaracidal virulent factors during pathogenesis to ticks.

INTRODUCTION

Ticks are considered major vectors of animal diseases and are second only to mosquitoes as vectors of human infectious diseases. Fungi play a major role in the ecological systems of ticks. Beauveria bassiana is an entomopathogenic fungus with demonstrated virulence towards a large range of insect hosts and is considered to be a facultative insect pathogen (Bidochka et al., 2002). B. bassiana has been found to be associated with pathogenicity to ticks of the Ixodidae family, including Ixodes scapularis Say, Boophilus decoloratus Koch, Rhipicephalus appendiculatus Neumann, Amblyomma variegatum Fabricius, and A. maculatum Koch (Chandler et al. 2000, Kaaya 2000, Samish 2000, Benjamin et al. 2002, Fernandes et al. 2003). These studies have highlighted the potential for B. Bassiana in tick control.

Little to no mortality was observed using B. bassiana fungal conidia or blastospores washed into dH2O, buffer, or fresh media, but greater than 50% mortality within 14-28 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. Our studies have implicated oxalic acid (OA) as the acaracidal virulence factor in B. bassiana.

Oxalic acid has been detected in various organisms, including animals, plants and fungi. Certain plant pathogenic fungi secrete oxalic acid as part of the process for invasion of plant tissues. For example, Sclerotium rolfsii Sacc., a fungus, causes diseases of plants in nearly 100 plant families. During infection, the fungus produces high levels of a necrosis phytotoxin identified as oxalic acid (Noyes 1981). Oxalic acid may have a number of functions in the infection process including chelating calcium from the cell thus making the pectic fraction more available to fungal hydrolases, and providing an acid pH needed for maximum activity of the wall degrading enzymes released by the pathogenic fungus (Keates et al, 1996, Caliskan 2000).

OA production in fungi is often related to metal detoxification and pathogenesis; the latter due to affects that include acidification 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).

Oxalate crystals have been identified on cadavers of insects infected by fungi, including B. bassiana (Moino et al. 2002). Metabolic acids, including oxalate, have been implicated in B. bassiana mediated virulence towards the migratory grasshopper, Melanoplus sanguinipes Fabricius (Bidochka and Khachatourians 1991). In this study, we report that oxalic acid display a pH dependent toxicity towards 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.

BACKGROUND

Fungus as biological Control

Entomogenous fungi have been used successfully to control various agricultural and pasture pests. In Brazil, they have been sprayed with airplanes in large fields to control sugarcane pests (Gillespie and Claydon, 1989), whereas in Indonesia and Malaysia, they are used to control the rhinoceros beetle, a serious pest of oil palms (Munaan and Wikardi, 1986). There are several advantages to using fungi as pest control agents, because they have little or no environmental effects (naturally occurring) and they have a low potential health hazard for vertebrates (McCoy, 1988).

Beauveria bassiana as a biological control for ticks

Ticks are the most harmful ectoparasites of domestic and wild animals as well as important vectors of disease agents to humans. A high mortality and reduced fecundity and egg viability has been reported in ticks experimentally-infected with the entomogenous fungi, B. bassiana and M. anisopliaie (Kaaya et al., 1996). There is a differential response to the fungus depending on the fungal and tick species. It has been reported that tick species display differential susceptibility to the entomopathogenic fungi B. bassiana and M. anisopliase and that the ability to overcome fungistatic compounds present in the tick epicuticle may determine the likelihood of successful infections and virulence (Keyhani et al., 2004).

Oxalic acid production in fungus

Oxalic acid is one of the strongest organic acids with pKa values of 1.3 and 4.3. Oxalic acid has been detected in various organisms, including animals, plants and fungi. Oxalic acid is produced by a wide variety of fungi, including brown-rots, white-rots, mycorrhizae, plant pathogens, and Aspergillus niger. The biochemical metabolic pathway of Beauveria bassiana has not yet been fully investigated. In theory the oxalate production would be similar to that of the filamentous fungus A. niger. In A. niger, oxalate biosynthesis is due exclusively to the action of oxaloacetase, which catalyzes the hydrolysis of oxaloacetate to oxalate and acetate (Hayaishi et al., 1956; Mueller, 1975; Lenz et al., 1976; Kubicek et al., 1988). The enzyme is located in the cytosol, and expression is induced at pH values greater than 4 and by carbonate (Kubicek et al., 1988; Pedersen et al., 2000b). The enzyme requires Mn²⁺ or activity (Km = 21 µM), which has obvious implications for the citric acid process, and is specific for oxaloacetate (K_m =220 µM) (Hayaishi et al., 1956; Lenz et al., 1976). The known presence of pyruvate carboxylase in the cytosol of A. niger (Osmani and Scrutton, 1983; Bercovitz et al., 1990; Jaklitsch et al., 1991) together with the insensitivity of oxalate production to the TCA cycle inhibitor fluorocitrate (Kubicek et al., 1988) indicates that oxalate is produced by a branch from the glycolytic pathway (as shown in Fig. 1).

Figure 1. Critical pathways for organic acid synthesis in Aspergillus spp. Numbers refer to the proteins and genes listed in Table 1. Abbreviations: PPP, pentose phosphate pathway; Glc, glucose; Fru, fructose; Suc, sucrose; Tre, trehalose; GlcA, gluconic acid; P, phosphate; DiP, diphosphate; Glyc, glyceraldehydes; DHAP, dihydroxyacetone phosphate; GlycA, glyceric acid; PEP, phosphoenolpyruvate; OAA, oxaloacetic acid; Mal, malic acid; Acon, aconitic acid. (Magnuson and Lasure, 2004).

Oxalic acid analysis using high performance liquid chromatography (HPLC)

HPLC is a popular method of analysis because it is easy to learn and use and is not limited by the volatility or stability of the sample compound. The analysis of the oxalic acid present daily during the growth of the cultures is analyzed via HPLC. Quantification of compounds by HPLC is the process of determining the unknown concentration of a compound in a known solution. It involves injecting a series of known concentrations of the standard compound solution onto the HPLC for detection. The chromatograph of these known concentrations will give a series of peaks that correlate to the concentration of the compound injected. Calibrating for these known concentrations will enable the detector to calculate the concentration of the unknown solutions.

MATERIALS AND METHODS

Fungal strain: Maintenance and Growth

Beauveria bassiana (ATCC 30517) was grown on Potato dextrose agar (PDA) or on Sabouraud dextrose + 0.5% yeast extract on either agar plates (SDAY) or in liquid broth (SDY). Plates were incubated at 26°C for 10-12 days and conidia were harvested by flooding the plate with sterile distilled water containing 0.01% Tween-20. Conidial suspensions were filtered through glass wool and final spore concentrations were determined by direct count using a hemocytometer. 50 milliliter liquid broth cultures of various media including potato dextrose (PD), sabouraud dextrose (SAB), glucose (Glc), n-acetyl glucosamide (Glc Nac), Yeast (YE), Czapek-Dox (CZ) all with 0.5% Yeast and without 0.5% yeast were inoculated with conidia harvested from plates to a final concentration of 5 x 10⁵ conidia/ml. Cultures were grown for 14-17 days at 26°C with aeration.

Sampling

Daily samples were taken from the growing cultures in the different media. Under sterile conditions 1600_l of the fungal mass cell from each culture was taken and centrifuged at 10000xg for 15 minutes. The cell culture supernatant (spent media) was filtered through a 0.22 mm sterilization membrane. Aliquots of the spent culture supernatant were stored either at 4°C or -20°C.

Treatment of ticks

Ticks were submerged for ~ 60 sec in experimental solutions (e.g. spent culture supernantant, 1-50 mM oxalate, pH 4.0 & pH 7.0, etc) and the excess suspension removed with either a pipet or a cotton swab. Each trial included 20-50 ticks, and trials at each concentration replicated 3 times. Ticks were placed in conical tubes or microtiter plates containing numerous needle-puncture holes (to allow for free-flow of air exchange) and stoppered with styrofoam plugs. Specimens were placed in a humidity chamber (>90% RH) with a 12 hr day (27°C)/night (25°C) cycle, with mortality recorded every day.

Sample preparation

Preparing the samples for High performance Liquid Chromatography (HPLC) analysis. 0.7ml of the supernatant obtained was filtered through a 5,000 molecular weight cut-off membrane (VivaScience) and the filtrate was acidified by addition of 5_l of dilute 1N sulfuric acid before injection (10-20 μl) onto the HPLC column. The acidification was to dissolve any cell debris that may still be present.

Oxalic acid HPLC analysis

The organic acid and carbohydrate concentrations were analyzed by high-performance liquid chromatography (Hewlett Packard 1090 series II chromatograph equipped with refractive index and UV₂₁₀ detectors) with a Bio-Rad Aminex HPX-87H ion exclusion column run isocratically in 4mM H₂SO₄. Each sample was run under a method for 40 minutes at a flow rate of 4ml/min of 4 mM H₂SO₄ at 45°C at a pressure of 60 barr. The chromatographic profiles of oxalic acid and other acids were detected by ultra violet absorbance. Oxalic acid peaks were identified by the retention time of an oxalic acid standard obtained at different concentrations of the standard.

Mutagenesis

Chemical mutants of B. bassiana strain 30517 were produced using the alkylating reagent ethyl methanesulfonate (EMS). A spore suspension (0.1 ml of 1-5 x 10⁷ conidia/ml) was added to 0.9 ml potassium phosphate buffer (50 mM, pH 7.0) containing 10 μl EMS. Cells were incubated at 26°C for 5-8 h with aeration. Samples (0.1-0.5 ml) were diluted 1:10 into buffer containing 10% (w/v) sodium thiosulfate and incubated for an additional 0.5-1 h with aeration. Samples were diluted (typically to 0.5-1 x 10³ 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 8-10 d. Colonies that lacked, or had increased zones of yellow (pH 6.8 plates) were removed, single spore isolated, and rescreened on solid pH-indicator media over several generations (3-5 times).

RESULTS

Ticks exposed to supernatant

Unfed adult A. americanum were susceptible to cell-free culture supernatants derived from growth of the entomopathogenic fungi Beauveria bassiana in different media (Table 2). High mortality was observed within 14 days using fungal spent media derived 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 65-85% (total) mortality within 28 d of the original treatment, whereas 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 50 ± 10% and 32 ± 15% (14 d post-treatment) mortality, respectively. Control treatments with sterile media or dH₂O resulted in less than 5% mortality throughout the time course of the experiments. In order to test the heat lability of the observed acaracidal activity, aliquots of SD or SDY 6 d culture supernatants boiled for 10 min, allowed to cool to room temperature for 10 min, briefly 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. Similarly, aliquots of SD and SDY culture supernatants treated with proteinase K (1mg/ml) for 30 min at 37°C resulted in 24 ± 6 % and 50 ± 10 % mortality (14 d post-treatment). By contrast, dialysis using a 10,000 molecular weight cut-off membrane of active culture supernatants against 50 mM Tris buffer, pH 7.0 resulted in marked reduction in the acaracidal potency of the culture supernatants; with total mortality percentages dropping to 5 ± 3 % for SD and 9 ± 3 % for SDY media, 14 d post-treatment of adult A. americanum ticks. These data pointed towards the likely existence of small heat-stable acaracidal compound(s) secreted by B. bassiana into SD and SDY media during growth.

Presence of oxalic acid

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 2). The concentration of oxalic acid and the resultant decrease in pH correlated with the acaracidal activity of the culture supernatants. High concentrations of oxalate (20-35 mM) were produced when the fungal cells were grown in SD or SDY media, whereas lower 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.

Effects of oxalic acid

The acaracidal toxicity of oxalate was tested by using the chemical compound to treat adult A. americanum ticks (Fig.2). Greater than 60% (14 d post treatment) and approximately 20% mortality was observed using 50 and 20 mM oxalate, pH 4.0, respectively. No significant mortality was observed using a single treatment with lower concentrations of oxalate (1-10 mM), pH 4.0, or with solutions of oxalate at pH 7.0 (1-50 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. 2). A second treatment 21 d after the initial treatment resulted in 75-85 % total mortality using either 20 or 50 mM oxalate, pH 4.0, and approximately 40% mortality using 50 mM oxalate, pH 7.0 within 14 d (35 d total).

Figure 2. 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, 50 mM oxalate pH 7.0 (_), all other conditions tested including 1-50 mM citrate at pH 4.0 and 7.0, 1-50 mM formate at pH 4.0 and 7.0, 1-10 mM oxalate pH 4.0, and 1-20 mM oxalate pH 7.0. Values given are means of three experiments ± SE.

Oxalate activity and pH dependance

The pH dependence of the acaracidal activity of oxalate was investigated using oxalic acid solutions ranging in pH values from 4.0-7.0 (Fig. 3). Acaracidal activity was highest at a pH value less that 4.0 (> 80 % mortality, 14 d post treatment) with tick mortality rapidly decreasing as the pH of the oxalate solution was raised. Mortality at pH 4.5 was 4-fold lower than that observed for solutions of oxalate at pH 4.0. Second treatments, 14 d after the first, had a minor effect, resulting in up to 40% total mortality in some instances.

Figure 3. 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 (◊), mortality 14 d later, after a second treatment on day 14 (28 d total, _). Values given are means of at least three experiments ± SE.

Chemical mutants

In order to assess whether oxalate production by B. bassiana was indeed a contributing factor in the acaracidal activity of culture supernatants, mutant screens were established in order to isolate oxalate non-producers. Because oxalate is one of the secreted organic acids responsible for the acidification of the media, surviving colonies of EMS-treated conidia were plated onto SDYA media supplemented with the pH indicator dye bromocresol purple (pH_initial = 6.8). Approximately 5,000 mutant clones of B. bassiana strain 30175 were screened on the indicator plates and 6 mutants were identified that lacked or had reduced zones of surrounding yellow color (acidification). Of these, three appeared to be false positives and produced wild type yellow zones when single spore isolated and rescreened. The remaining three mutants, designated as clones A1+15, A1+16, and A1+17, appeared to retain their phenotype (no yellow zone of clearing, purple colonies) after at least three generations of re-screening on the indicator plates. Mutants A+15 and A1+16 displayed altered conidiation affects, 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 on under any of the conditions tested. The concentration of oxalate secreted by the mutants was quantified over a 15 d time course of growth in SDY broth (Fig. 4). 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 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 previously described. Less than 10% mortality towards 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. 4. Concentration of oxalate secreted into the medium during growth in SDY broth, wild-type B. bassiana (_), mutants A+15 and A+16 (_), and mutant A+17 (_).

Nutrient conditions affect oxalate production

Oxalic acid production of B. bassiana grown in the liquid broths of potato dextrose (PDA), Sabouraud Dextrose (SD), Czapek-Dox (CzD), Glucose (Glc), N-acetyl glucosamide (GlcNac), Glucose + 0.5%Yeast extract (GlcYE), N-acetyl glucosamide 0.5%Yeast extract (GlcNacYE) was analyzed by HPLC. When enriched with yeast higher concentrations of oxalic acid were produced in GlcYE and GlcNacYE peaking at concentrations of 10.9mM and 24.6mM respectively compared to Glc and GlcNac, which produce 7.0mM and 0.7mM respectively by day 5 (Fig. 5). PDA, SD, and YE (Fig. 6) are considered to be nutrient rich media which produce higher concentrations of oxalic acid within 3-6 days of growth, whereas CzD, Glc, GlcNac (Fig. 7) are considered minimal media, having lesser nutrients comparatively and yield lower concentrations of oxalic acid at the 3-6 day period.

Figure 5. Variance in production of oxalic acid with and without the addition of 0.5% yeast extract, Glucose (_), N-acetyl glucosamine (--_--), Glucose + 0.5%Yeast extract (_), and N-acetyl glucosamine + 0.5%Yeast extract (--x--).

Figure 6. Oxalic acid production in rich media, potato dextrose (_), Sabouraud Dextrose (_), and Yeast extract (_).

Figure 7. Oxalic acid production in minimal media, Glucose (_), N-acetyl glucosamine (--_--), and Czapech-Dox (_).

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 towards several insect species including the tarnished plant bug Lygus hesperus (Alverson 2003) and the migratory grasshopper Melanoplus sanguinipes (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.

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 towards these ticks (and others) depending upon the fungal growth and media composition. HPLC analysis of culture supernatants coupled to tick mortality experiments confirmed that one of the major acaracidal 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 contribute to the establishment and progression of the disease.

Despite its 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 values greater than 4.5 (single application). These data suggest that (as a diprotic compound with pK_as 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 in promoting pathogenesis. Oxalate concentrations in culture supernatants did approach 30-35 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) and this is likely to increase their toxicity. Indeed, oxalic acid is able to solubilize several components of insect cuticles including elastin, 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 less than 1% of the wild-type levels of oxalate under the conditions tested). Culture supernatants derived from all three mutants were non-toxic towards A. americanum ticks. While these observations support the hypothesis that oxalate is an important fungal virulence factor during pathogenesis towards ticks, some caution should be taken in interpreting these results. 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 were also deficient 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) due to the fact that these clones likely contain multiple mutations that would have obscured interpretation of any results, particularly since the mutants displayed altered developmental and conidiation phenotypes. Future research using targeted-gene knockouts of enzymes in the oxalate biosynthetic pathway(s) of B. bassiana will likely help shed light on the physiological role of oxalate during pathogenesis.

In varying nutrient media or upon addition of nutrient rich yeast extract in minimal media, a clear correlation is evident; the production of oxalic acid in B. bassiana depends on growth and nutrient conditions. Finally, our results indicate that examining inoculum conditions that would favor oxalate production could increase the efficacy of field applications of B. bassiana in biocontrol efforts. This 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.

---

ACKNOWLEDGEMENTS

I would like to thank Dr. Nemat Keyhani for his guidance and assistance throughout my research and Brett Kirkland for his help and contribution to this paper.

---

REFERENCES

1. Alverson, J. 2003. Effects of mycotoxins, kojic acid and oxalic acid, on biological fitness of Lygus hesperus (Heteroptera: Miridae). J Invertebr Pathol 83: 60-2.
   
2. Balmforth, A. J., and A. Thomson. 1984. Isolation and characterization of glyoxylate dehydrogenase from the fungus Sclerotium rolfsii. Biochem J 218: 113-8.
   
3. Bateman, D. F., and S. V. Beer. 1965. Simulanteous production and synergistic action of oxalic acid and polygalacturonase during pathogenesis by Sclerotium rolfsii. Phytopathology 58: 204-211.
   
4. Benjamin, M. A., E. Zhioua, and R. S. Ostfeld. 2002. Laboratory and field evaluation of the entomopathogenic fungus Metarhizium anisopliae (Deuteromycetes) for controlling questing adult Ixodes scapularis (Acari: Ixodidae). J Med Entomol 39: 723-8.
   
5. Bidochka MJ, Menzies FV, Kamp AM. 2002. Genetic groups of the insect-pathogenic fungus Beauveria bassiana are associated with habitat and thermal growth preferences. Arch Microbiol. Dec;178(6):531-7. Epub 2002 Oct 08.
   
6. Bidochka, M. J., and G. G. Khachatourians. 1991. The implication of metabolic acids produced by Beauveria bassiana in pathogenesis of the migratory grasshopper, Melanoplus sanguinipes. J Invertebr Pathol 58: 106-117.
   
7. Caliskan, Mahmut. 2000. The metabolism of oxalic acid. Turk J Zool 24: 103-106.
   
8. Cessna, S. G., V. E. Sears, M. B. Dickman, and P. S. Low. 2000. Oxalic acid, a pathogenicity factor for Sclerotinia sclerotiorum, suppresses the oxidative burst of the host plant. Plant Cell 12: 2191-200.
   
9. Chandler, D., G. Davidson, J. K. Pell, B. V. Ball, K. E. Shaw, and K. D. Sunderland. 2000. Fungal Biocontrol of Acari. Biocon. Sci. Tech 10: 357-384.
   
10. Eisler, M. C., S. J. Torr, P. G. Coleman, N. Machila, and J. F. Morton. 2003. Integrated control of vector-borne diseases of livestock--pyrethroids: panacea or poison? Trends Parasitol 19: 341-5.
    
11. Fernandes, E. K., G. L. Da Costa, E. J. De Souza, A. M. De Moraes, and V. R. Bittencourt. 2003. Beauveria bassiana isolated from engorged females and tested against eggs and larvae of Boophilus microplus (Acari: Ixodidae). J Basic Microbiol 43: 393-8.
    
12. Franceschi, V. R., and H. T. Horner. 1980. Calcium oxalate crystals in plants. Bot Rev 46: 361-427.
    
13. Gadd, G. M. 1999. Fungal production of citric and oxalic acid: importance in metal speciation, physiology and biogeochemical processes. Adv Microb Physiol 41: 47-92.
    
14. George, J. E. 2000. Present and future technologies for tick control. Ann N Y Acad Sci 916: 583-8.
    
15. Gillespie, A.T. and Claydon, N. 1989.The use of entomogenous fungi for pest control and the role of toxins in pathogenesis. Pesticide Sci. 27: 203–215
    
16. Godoy, G., J. R. Steadman, M. B. Dickman, and R. Dam. 1990. Use of mutants to demonstrate the role of oxalic acid in pathogenicity of Sclerotinia sclerotiorum on Phaseolus vulgaris. Phys. Mol. Plant Path. 37: 179-191.
    
17. Gregorc, A., and J. Poklukar. 2003. Rotenone and oxalic acid as alternative acaricidal treatments for Varroa destructor in honeybee colonies. Vet Parasitol 111: 351-60.
    
18. Jarosz-Wilkolazka, A., and G. M. Gadd. 2003. Oxalate production by wood-rotting fungi growing in toxic metal-amended medium. Chemosphere 52: 541-7.
    
19. Kaaya, G. P. 2000. Laboratory and field evaluation of entomogenous fungi for tick control. Ann N Y Acad Sci 916: 559-64.
    
20. Kaaya, G.P., Mwangi, E.N. and Ouna, E.A. 1996. Prospects for biological control of livestock ticks, Rhipicephalus appendiculatus and Amblyomma variegatum using the entomogenous fungi Beauveria bassiana and Metarhizium anisopliae. J. Invert. Pathol. 67: 15–20
    
21. Kanga, L. H., R. R. James, and D. G. Boucias. 2002. Hirsutella thompsonii and Metarhizium anisopliae as potential microbial control agents of Varroa destructor, a honey bee parasite. J Invertebr Pathol 81: 175-84.
    
22. Kanga, L. H., W. A. Jones, and R. R. James. 2003. Field trials using the fungal pathogen, Metarhizium anisopliae (Deuteromycetes: Hyphomycetes) to control the ectoparasitic mite, Varroa destructor (Acari: Varroidae) in honey bee, Apis mellifera (Hymenoptera: Apidae) colonies. J Econ Entomol 96: 1091-9.
    
23. Keates, S.A., Zhang, D., Loewus, F.A. and Franceschi, V.R. 1996. Oxalate oxidase is synthesized and secreted from bean leaf cells in response to fungal infection. Plant Physiol. 111, p.311.
    
24. Kirkland, B. H., G. S. Westwood, and N. O. Keyhani. 2004a. Pathogenicity of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to Ixodidae tick species Dermacentor variabilis, Rhipicephalus sanguineus, and Ixodes scapularis. J Med Entomol 41: 705-11.
    
25. Kirkland, B. H., E.-M. Cho, and N. O. Keyhani. 2004b. Differential Susceptibility of Amblyomma maculatum and Amblyomma americanum (Acari:Ixodidea) to the Entomopathogenic Fungi Beauveria bassiana and Metarhizium anisopliae. Biological Control 31: 414-421.
    
26. Kirkland, B. H., A. N. Eisa, N. O. Keyhani. 2004c. Oxalic acid as a fungal acaracidal virulence factor. (submitted for publication)
    
27. Kubicek, C. P. 1987. The role of the citric acid cycle in fungal organic acid fermentations. Biochem Soc Symp 54: 113-26.
    
28. Kubicek, C. P., G. Schreferl-Kunar, W. Wohrer, and M. Rohr. 1988. Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger. Appl Environ Microbiol 54: 633-7.
    
29. Magnuson Jon K. and Linda L. Lasure, 2004. Organic Acid Production by Filamentous Fungi Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine. Edited by Jan and Lene Lange, Kluwer Academic/Plenum Publishers.
    
30. Magro, P., P. Marciano, and P. Di Lenna. 1988. Enzymatic oxalate decarboxylation in isolates of Sclerotinia sclerotiorum. FEMS Microbiol Lett 49: 49-52.
    
31. Maxwell, D. P., and D. F. Bateman. 1968. Oxalic acid biosynthesis by Sclerotium rolfsii. Phytopathology 58: 1635-1642.
    
32. McConn, M. M., and P. A. Nakata. 2002. Calcium oxalate crystal morphology mutants from Medicago truncatula. Planta 215: 380-6.
    
33. McCoy, C.W., R.A. Samson and D. 1998. Boucias, Entomogenous fungi, in Handbook of Natural Pesticides, C.M. Ignoffo and N. B. Mandava (eds). CRC Press: Boca Raton, FL. P. 151-236.
    
34. Moino, A., S. B. Alves, R. B. Lopes, P. M. Oliveira, J. Neves, R. M. Pereira, and S. A. Vieira. 2002. External development of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae in the subterranean termite Heterotermes tenuis. Sci. Agri. 59: 267-273.
    
35. Munaan, A. and Wikardi, E.A. 1986. Towards the biological control of coconut insect pests in Indonesia. In: Biological Control in the Tropics, M.Y. Hussein, and A.G. Ibrahim (eds), pp. 149–157, Penerbit Universiti Partanian, Malaysia, Serdang
    
36. Munir, E., J. J. Yoon, T. Tokimatsu, T. Hattori, and M. Shimada. 2001. A physiological role for oxalic acid biosynthesis in the wood-rotting basidiomycete Fomitopsis palustris. Proc Natl Acad Sci U S A 98: 11126-30.
    
37. Noyes, R.D. and Hancock, J.G. (1981) Role of oxalic acid in the Sclerotinia wilt of sunflower. Physiol. Plant Pathol. 18. 123-132
    
38. Oke, O. L. 1969. Oxalic acid in plants and in nutrition. World Rev Nutr Diet 10: 262-303.
    
39. Pegram, R. G., D. D. Wilson, and J. W. Hansen. 2000. Past and present national tick control programs. Why they succeed or fail. Ann N Y Acad Sci 916: 546-54.
    
40. Rollins, J. A., and M. B. Dickman. 2001. pH signaling in Sclerotinia sclerotiorum: identification of a pacC/RIM1 homolog. Appl Environ Microbiol 67: 75-81.
    
41. Samish, M. 2000. Biocontrol of ticks. Ann N Y Acad Sci 916: 172-8.
    
42. Sonenshine, D. E. 1993. Biology of Ticks. Oxford University Press, New York.
    
43. St Leger, R. J., J. O. Nelson, and S. E. Screen. 1999. The entomopathogenic fungus Metarhizium anisopliae alters ambient pH, allowing extracellular protease production and activity. Microbiology 145 ( Pt 10): 2691-9.

---

--top--

Back to the Journal of Undergraduate Research