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The implication of metabolic acids produced by Beauveria bassiana in pathogenesis of the migratory grasshopper, Melanoplus sanguinipes
JOURNAL
OF INVERTEBRATE
PATHOLOGY
58,
106-117 (1991)
The Implication of Metabolic Acids Produced by Beauveria in Pathogenesis of the Migratory Grasshopper, Melanoplus MICHAEL Bioinsecticide
J. BIDOCHKA
Research Laboratory, University of Saskatchewan,
bassiana
sanguinipes
AND GEORGE Department Saskatoon,
G. KHACHATOURIANS’
of Applied Microbiology Saskatchewan, Canada
and Food Science, S7N OWO
Received May 23. 1990; accepted September 25, 1990 The entomopathogenic fungus, Beauveria bassiana, produced oxalic and citric acids in liquid cultures containing grasshopper (Mefanoplus sanguinipes) cuticle as the sole nutrient source. Oxalic and citric acids solubilized cuticular proteins as well as commercial preparations of elastin and collagen. Other organic acids which solubilized cuticular proteins included acetic and formic acids. In contrast to these organic acids, the inorganic acids, hydrochloric acid and sulfuric acid, did not solubilize or only slightly solubilized cuticular proteins when compared to the organic acids. M. sanguinipes treated with B. bassiana showed a LT,, of 7.33 days, while M. sanguinipes treated with citric acid or oxalic acid showed a LT,, of 7.25 and 13.28 days, respectively. M. sanguinipes treated with citric acid followed by a B. bassiana conidia treatment showed a LT,, of 3.88 days. and the oxalic acid. B. bassiana treatment resulted in a LT,, of 5.08 days. Analysis of the bioassay data revealed that the relationship between oxalic acid or citric acid together with B. bassiana conidia in grasshopper mortality was markedly synergistic. We suggest that acid metabolites produced by B. bassiana may play a role in cuticle solubihzation and subsequent hyphal penetration. 0 1991 Academic Press. Inc. KEY WORDS: Beauveria bassiana; Melanoplus sanguinipes: migratory grasshopper; metabolic acids; oxalic acid; citric acid: pathogenesis.
INTRODUCTiON
The initial stages of insect infection by entomopathogenic fungi include the penetration of the host cuticle. Several studies have shown that penetration of the cuticle is accomplished by the action of fungal extracellular enzymes accompanied by mechanical pressure of the advancing hyphae. For example, extracellular protease produced by Beauveria bassiana (Bidochka and Khachatourians, 1987, 1990) and Metarhizium anisopliae (St. Leger et al., 1988) are important determinants of fungal virulence toward the migratory grasshopper, Melanoplus sanguinipes, and the tobacco hornworm. Manduca sexta, respectively. Chitinase produced by Nomuraea rileyi is an important enzyme in pathogenicity toward Trichoplusia ni (El-Sayed et al., 1989). Mechanical pressure is also associated with M. anisopliae penetration of M. ’ To whom reprint requests should be addressed.
sexta procuticle
(Goettel et al., 1989). Other factors such as fungal metabolic acids which may contribute in the solubilization or hydrolysis of insect cuticle have only been speculated upon (Charnley, 1984). but experimental evidence is lacking. Many species of fungi produce an assortment of metabolites, including various organic acids. Of the entomopathogenic fungi, Beauveria tenella produces oxalic acid (Cordon and Schwarz, 1962) and oxalic acid-like crystals have been observed on the surface of insects infected by B. bassiana (Dresner, 1950). Oxalic acid solubilizes elastin (Kokoglu et al., 1978; Partridge et al., 1955; Adair et al., 1951) and elastinlike (i.e., resilin) tissue comprises part of the cuticle of certain insects (Andersen and Weis-Fogh, 1964). For analytic purposes, dilute acids, including oxalic acid, have been used to partially hydrolyze proteins into smaller peptides for further determination of the amino acid composition (Maravalhas, 1970). In fact, the use of dilute 106
0022-201 l/91 $1.50 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
B. bassiana
METABOLIC
acids is one of the oldest and simplest chemical methods for fragmenting protein chains (Partridge and Davis, 1950). In the present study we confirm the production of oxalic acid by B. bassiana and present the first report on the production of citric acid by this fungus in liquid cultures containing insect cuticle as the sole source of nutrients. Citric acid was studied since oxalic acid is a by-product of citric acid formation in fungi (Crueger and Crueger, 1984). The effects of these metabolic acids and other acids on the solubilization of cutitular proteins of the migratory grasshopper, M. sanguinipes, are reported. Finally, the possible synergistic role of metabolic acids during pathogenesis of B. bassiana is examined by pretreating M. sanguinipes with dilute acid solutions and then conducting a bioassay using B. bassiana. This is the first such study which investigates the possible role of metabolic acids produced by this entomopathogenic fungus in the insect infection process. MATERIALS
AND METHODS
Preparation of B. bassiana conidia and rearing conditions of the migratory grasshopper, M. sanguinipes, were described previously (Bidochka and Khachatourians, 1990). The insoluble proteins, elastinorcein and analytical grade elastin, were prepared from bovine neck ligament, and collagen was prepared from bovine achilles tendon (Sigma Chemical Co., St. Louis, Missouri). The acids which were used were of reagent grade and were diluted with sterile-distilled water to yield 0.1 mM to 1 M solutions. Preparation of ground M. sanguinipes cuticle. The method of Andersen (1980) for the bulk preparation of M. sanguinipes cuticle was used. Frozen grasshoppers, which had previously been starved for 24 hr, were homogenized in a Waring blender containing 1% (w/v) potassium tetraborate (100 grasshoppers, of equal sex ratio, per liter solution). The material was collected from
ACIDS
107
a plastic sieve (l-mm screen), dried, and milled to a tine powder (
Ox-
alic acid production and citric acid production by B. bassiana grown in liquid cultures were determined by enzymatic analysis using Boehringer Mannheim test kits (Federal Republic of Germany). Insoluble proteins and grasshopper cuticle treatment with acids. The ability of var-
108
BIDOCHKA
AND
ious acids to solubilize the following insoluble proteins was tested: elastin-orcein, analytical grade elastin, collagen, or the protein components of M. sanguinipes powdered cuticle. The structural proteins, collagen and elastin, are insoluble in distilled water. However, peptide components of these proteins may be solubilized if the intact protein is treated with certain acids. Ten milligrams of one of these insoluble proteins or M. sanguinipes powdered cuticle was incubated in 2 ml of 0.1 mM to 1 M organic acid (oxalic, citric, boric, or formic acid) solutions. Likewise ammonium and sodium salts of oxalic and citric acids were also tested. The suspensions were incubated at 27°C for 24 hr, then passed through membrane filters (HA-type, 0.45-kmpore-size). Solubilization of elastin-orcein was measured as the release of a chromophore which absorbs at 520 nm. Solubilization of analytic grade elastin, collagen, or powdered M. sanguinipes cuticle was measured as the release of soluble protein and free amino nitrogen.
KHACHATOURIANS
dipped in acid were not washed subsequent to the B. bassiana treatment. The 3-set acid dip is sufficient to wet the grasshoppers, they then appeared dry after 1 hr. One group was dipped into the conidial suspension only; this is the B. bassiana treatment. Of the control groups, one group was dipped in sterile-distilled water and one group was left untreated. The grasshoppers were individually housed and placed in an environmental chamber (27°C 30% relative humidity) and fed barley grass daily at which time the mortality was checked. Probit analysis was performed on Abbott corrected data (Abbott, 1925). Analysis of synergism between citric acid or oxalic acid and B. bassiana during the bioassay against M. sanguinipes was done using the “Dose-Effect Analysis with Microcomputers” software program (Elsevier Science Publ., The Netherlands) operated on an Apple Be computer. Scanning electron microscopy. Pieces of M. sanguinipes cuticle (approx 0.5 cm2) were excised from the abdomen. Carefully, Soluble protein and free amino nitrogen any loose tissue was scraped away without determination. Soluble protein was deterdamaging the cuticle. The cuticle was mined by the protein dye-binding assay of washed with sterile-distilled water and a Bradford (1976) using bovine y-globulin as streptomycin solution (100 pg/ml), then inwater, 1 mM hythe standard. Free amino nitrogen was de- cubated in sterile-distilled termined by the trinitrobenzene sulfonic drochloric acid, citric acid, or oxalic acid acid assay using glycine as the standard, for 1 hr. The cuticle was then placed on a and this assay was previously described by sterile Whatman No. 2 filter paper which Bidochka and Khachatourians (1990). had been soaked in water and placed in a Bioassay procedures. Adult M. sanguisterile Petri dish. Ten microliters of lo5 conipes were incapacitated with carbon dioxnidia/ml (approx lo3 conidia) was placed on ide. Each of the seven treatment groups the surface of the cuticle. The Petri dish and the two control groups consisted of 60 was sealed with parafilm and placed in an grasshoppers. Three groups of grasshopenvironmentally controlled chamber for 48 pers were dipped, for 3 set, into a 1 mM hr at 27°C. The cuticle pieces were then hydrochloric, oxalic, or citric acid solution, fixed in 2.5% glutaraldehyde in 10 mM porespectively. These are the acid treatments. tassium phosphate buffer (PH 7) for 24 hr, Three groups were dipped into one of the dehydrated in a series of ethanol solutions three acid solutions, then after 1 hr, each and then 100% acetone. The samples were grasshopper was dipped into a B. bassiana critical point dried in a Polaron drier and conidial suspension (10’ conidia/ml) for 3 then coated with gold in a Technics sec. These are the acid + B. bassiana treat“Hummer” sputter coater. Upper surfaces ments. The grasshoppers which were of the cuticle were viewed and photo-
B. bassiana
METABOLIC
graphed through a Cambridge Instrument Stereoscan Mark 2A scanning electron microscope operated at -20 kV. RESULTS Production of Metabolic Acids by B. bassiana Grown in Liquid Culture Containing Grasshopper Cuticle
Figure 1 shows the production of citric and oxalic acids by B. bassiana during growth on a 1% (w/v) suspension of M. sanguinipes powdered cuticle. Fifty percent of the conidia germinated within 24 hr. Oxalic acid production was low at day 2 (2.8 pg/ml) but increased at a rate of 50 pg oxalic acid/ml/day from days 2 to 3. Thereafter, oxalic acid levels remained at approximately 100 pg/ml. Similarly, citric acid showed three phases of production: (1) a low level of production up to day 3 at a rate of 7 kg/ml/day, (2) followed by an increase in the level of production from days 3 to 5 at a rate of 26 p.g/ml/day, and (3) and a decrease in citric acid levels up to day 6. Expressed in terms of molar equivalents at the highest levels of production, oxalic acid and citric acid levels were approximately 0.8 and 0.3 mM, respectively. Although the acid production increased with culture age the culture pH increased. This suggests that
8.5 8 7.5 I
-
2
0 Culture
4 Age
zl
6 (days)
1. Production of citric acid (0) and oxalic acid by Beauveria bassiana in liquid culture containing 1% (w/v) Melanoplus sanguinipes powdered cuticle. The pH (A) of the culture is also shown. Assays were performed in triplicate from duplicate cultures. SE bars are shown. FIG.
(0)
ACIDS
109
metabolic bases, possibly ammonia, were also being produced in cultures. Oxalic and citric acids were also formed in liquid media containing various combinations of 1% (w/v> gelatin, 1% (w/v) glucose, and 0.1 M CaCl,. There was no increase in oxalic acid or citric acid production in media containing CaCl,. We also checked if these acids originated from insect cuticle. Ten milligrams of cuticle was washed with 2 ml of 10 mM HCI in order to solubilize any calcium oxalate. We did not detect the presence of oxalic acid at levels of sensitivity of the assay (2 Fg/ml). Solubilization of Elastin and Grasshopper Cuticle by Various Acid
Table 1 shows the release of soluble products from elastin-orcein, elastin, collagen, and M. sanguinipes cuticle by various inorganic and organic acids or by distilled water alone. When compared with other acids, citric acid and oxalic acid showed the highest amount of elastin-orcein solubilization. Incubation of elastin-orcein in 1 M boric acid or distilled water resulted in the lowest solubilization of elastin-orcein. Since the soluble chromophore could be detected in the distilled water treatment, we suspect that a certain amount of watersoluble chromophore is nonspecifically released. No proteins were solubilized in water if the substrate was elastin or collagen. A small amount of protein was solubilized from elastin or collagen by the inorganic acids when compared with the organic acids. Of the organic acids, incubation of elastin with citric acid showed the highest release of soluble protein, and formic acid had the greatest activity on collagen. When the M. sanguinipes cuticle is suspended in distilled water, 2.6% of the dry weight of the cuticle was solubilized as protein. Since approximately 61% of the dry weight of M. sanguinipes cuticle is protein (Bidochka, 1989), the amount solubilized by distilled water represents about 4.2% of the total cutitular proteins by weight. Citric acid had the highest effect on solubilization of M.
110
SOLUBILIZATION
BIDOCHKA
OF ELASTIN-ORCEIN.
AND
KHACHATOURIANS
TABLE 1 ELASTIN, COLLAGEN. AND Melanoplus CONCENTRATIONS OF VARIOUS ACIDS
sanguinipes
CUTICLE
BY 1 M
Substrate’ Treatment Inorganic acid Hydrochloric Sulfuric Organic acid Acetic Boric Citric Formic Oxalic Water
Elastin-orcein
Elastin
Collagen
0.06 (0.01) 0.04 (0.02)
0.07 (0.01) 0
0.17 (0.03) 0.07 (0.02)
0.06 0.03 0.07 0.04 0.07
1.89 1.09 2.48 1.81 1.11
2.65 0.88 2.94 3.94 1.29
(0.01) (0.01) (0.01) (0.01) (0.02)
0.03 (0.02)
0
(0.17) (0.13) (0.07) (0.27) (0.07)
0
(0.24) (0.03) (0.10) (0.03) (0.20)
Cuticle
1.04 (0.18) 1.43 (0.37) 8.78 1.24 12.22 10.32 2.97
(1.87) (0.83) (0.66) (2.22) (0.69)
2.58 (0.24)
a Solubilization of elastin-orcein measured by release of a soluble chromophore detected at A,,,. Solubilization of elastin, collagen, and powdered M. sanguinipes cuticle was measured as the release of protein and expressed as the percentage (*SD) of the total dry weight from triplicate samples. The experiment was repeated twice with similar results.
cuticle. Approximately 12% of the dry weight of cuticle was solubilized as protein and this represents about 20% of the total cuticular proteins. Hydrochloric acid had the lowest amount of soluble protein release from cuticle. Of the organic acids, boric acid showed the lowest release of soluble protein from M. sanguinipes cuticle. The liberation of free amino nitrogen could not be detected in any of the samples in the range of sensitivity of the trinitrobenzene sulfonic acid assay. We were primarily interested in the effects of B. bassiana acid metabolites, especially oxalic and citric acids, on M. sanguinipes cuticle solubilization. We next examined the stoichiometric relationship between concentrations of oxalic acid and citric acid solutions and M. sanguinipes cuticle solubilization. Furthermore, we compared these effects to those of the salt forms of these acids, namely ammonium oxalate and sodium citrate, on cuticle solubilization. Figure 2A shows the release of soluble protein from M. sanguziupes cuticle by 0 to 1 M concentrations of oxalic acid or ammonium oxalate. The release of soluble proteins by the action of oxalic acid alone was highest at a 0.01 M concentration (Fig. 2A). sanguinipes
The release of soluble protein from M. sanguinipes cuticle decreased with oxalic acid concentrations greater or less than 0.1 M. It is interesting to note that the release of soluble protein by 1 M oxalic acid is only slightly higher than the effect of distilled water alone. This confirms the data in Table 1 which show that the release of soluble protein from M. sanguinipes cuticle by the action of 1 M oxalic acid solubilizes approximately 0.5% more protein when compared to the distilled water control treatment. On the other hand, the release of soluble protein from M. sanguinipes cuticle by the action of 0.01 M oxalic acid solubilizes approximately 5.5% more protein when compared to the distilled water control treatment. Figure 2B shows the release of soluble protein from M. sanguinipes cuticle by 0 to 1 M concentrations of citric acid or sodium citrate. Here the shape of the curve is quite different than that of the action of oxalic acid on the release of soluble protein from M. sanguinipes cuticle. The effect of citric acid on M. sanguinipes cuticle shows a saturation kinetics curve; a rapid increase of soluble protein from 0 to 0.1 M and a saturation of soluble protein release with citric
B. bassiana METABOLIC
ACIDS
111
values of 13.28 and 7.25 days. Hydrochloric acid treatment had no effect on M. sanguinipes when compared with the untreated controls. M. sanguinipes pretreated with hydrochloric acid followed by a B. bassi0.000 0.025 0.050 ana conidia treatment did not significantly reduce the LT,, values when compared to the B. bassiana conidia alone treatment. M. sanguinipes treated with oxalic or citric o!..,..r.‘,..n”,.‘, acid followed by a treatment with B. bassi0.0 0.2 0.4 0.6 0.8 1.0 1.2 ana conidia had lower LT,, values than the Oxalate [Ml singular treatments. There were no differences in M. sanguinipes mortality between the group which was dipped in steriledistilled water and the untreated group. The possibility of a synergistic effect between oxalic or citric acid and B. bassiana conidia on M. sanguinipes mortality was evaluated using a “Dose-Effect Analysis 0.025 0.050 with Microcomputers” software. Figure 3 00 shows the results of these analyses. The 0.0 0.2 0.4 0.6 0.8 1.0 1.2 combination index refers to the antagonisCitrate [Ml tic or synergistic interaction between two FIG. 2. Solubilization of Melanoplus sanguinipes treatments. A combination index greater cuticular proteins. Ten milligrams of powdered M. than 1 indicates that an interaction is antagsanguinipes cuticle was incubated in 2 ml of (A) oxalic acid (Cl) or ammonium oxalate (H) and (B) citric acid onistic; less than 1, the interaction is syn(0) or sodium citrate (W) solutions for 24 hr and sol- ergistic and an additive effect is indicated uble protein was measured. Insets show detailed reby a combination index equal to 1. The sults of protein solubilization for 0 to 0.05 M concenanalysis of the relationship between citric trations. Results are from triplicate assays. SE was acid and B. bassiana indicated that there less than 10% of the mean for each datum point. The experiment was repeated twice. was marked antagonism between citric acid and B. bassiana at low effect levels (i.e., ~20% of M. sanguinipes affected) but synacid concentrations greater than 0.1 M. No ergism at higher effect levels. The analysis free amino acids were detected as a result of the relationship between oxalic acid and of the action of citric or oxalic acid on M. B. bassiana indicated that there was slight sanguinipes cuticle. antagonism between citric acid and B. bassiana at effect levels less than 1% of M. Interactive Effects of Oxalic or Citric sanguinipes affected and markedly synerAcid and B. bassiana Pathogenesis gistic at higher effect levels. Therefore. at a low mortality of M. sanguinipes there is anTable 2 shows the summary for the probit tagonism between the oxalic or citric acids analyses for M. sanguinipes (i) treated with and B. bassiana. Another way the data may B. bassiana conidia, (ii) treated with 1mM oxalic, citric, or hydrochloric acids, and be interpreted is that 80% of the mortality (iii) pretreated with acid followed by a B. of M. sanguinipes in the B. bassiana-citric bassiana conidia treatment. The LTso for acid bioassay may be explained by the synM. sanguinipes treated with B. bassiana ergistic relationship between the two comof M. conidia was 7.33 days. Citric and oxalic ac- ponents. Likewise, 99% mortality ids also killed M. sanguinipes with LTso sanguinipes in the B. bassiana-xalic acid
112
BIDOCHKA
AND KHACHATOURIANS TABLE
PROBIT
ANALYSIS
DATA
FOR THE Beauveria
2
bassiana/AcrD-Melanoplus
sanguinipes
SYSTEM
Regression line equationd
LT,, fiducial limits’ Treatment”
Lower
LT,n
Upper
Intercept
Slope
conidia only Oxalic only Citric only HCl only* Oxalic + B. bassiana conidia Citric + B. bassiana conidia HCl + B. bassiana conidia
6.91 10.56 6.62 4.66 3.67 6.80
7.33 13.28 7.25 5.08 3.88 7.17
7.78 16.65 8.52 5.53 4.09 7.54
-1.54 0.29 1.21 -0.61 -3.67 -5.67
3.51 2.22 3.04 3.29 5.46 6.08
B. bassiana
a Oxalic, oxalic acid; citric, citric acid; HCl, hydrochloric acid. b LT,, values could not be calculated for the HCl-treated M. sanguinipes since no mortality differences could be observed when compared with the controls. ’ Fiducial limits and times in days (P = 0.05). d Equation of the best-fitting line using the maximum-likelihood method. Sample size for each treatment was 60 grasshoppers and the experiment was repeated once with similar results.
bioassay may be explained by the synergistic relationship between the two components. On the whole, the analyses indicated a marked synergistic action between oxalic acid or citric acid with B. bassiana. Effect of pH and the Associated Bases of Oxalic, Citric, or Hydrochloric Acid on B. bassiana Germination
The above date show that one of the possible factors responsible for the synergism between oxalic and citric acid and B. bassiana is the role of acids in solubilizing M. sanguinipes cuticle (Table 1, Fig. 2). Oxalic
citric
acid
oxalic
0 0.0 Fraction
"I.'I'.I"I..l 0.2
acid
0.4 of
t
LM
Bb t Bb
0.6 sanguinipes
0.8
1.0 affected
FIG. 3. The analysis of the interaction of citric acid plus Beauveria bassiana (Bb) or oxalic acid plus Bb in a bioassay against Melanoplus sanguinipes. The combination index refers to antagonism (>l), additive effects (= 1). or synergism (Cl).
or citric acid may loosen the cuticular proteins to facilitate hyphal penetration. Another possible explanation for the synergism between oxalic and citric acid and B. bassiana is the role of pH or the associated base on the germination B. bassiana conidia. The organic acid may lower the pH and the associated base may act as a nutrient source to facilitate conidial germination. To test this hypothesis, B. bassiana conidia were incubated in a 1% (w/v) M. sanguinipes cuticle medium with varying pHs adjusted with 1, 10, or 100 mM oxalic, citric, or hydrochloric acid and conidial germination was counted after a 24-hr incubation. Hydrochloric acid was included as the control inorganic acid. Figure 4 shows the results of this experiment. No differences in percentage of B. bassiana conidia germination were observed in the M. sanguinipes cuticle suspensions adjusted to approximately similar pH and similar molarity. Therefore, conidial germination is pH dependent but not dependent on the corresponding bases of oxalic, citric, or hydrochloric acid. SEM of Acid-Treated Grasshopper Cuticle and B. bassiana Penetration
Figure 5 shows some examples of B. bas-
B. bassiuna Molarity of 0.01 --0.1
o! 1
METABOLIC
Acids 0.001 -
I
I.
I
I.
I
,
2
3
4
5
6
7
PH
4. The percentage of Beauveriu bassiuna conidia germinating after 24 hr in liquid medium containing 1% (w/v) Melunoplus sanguinipes cuticle with 0.1, 0.01, or 0.001 M oxalic acid (O), citric acid (0), or hydrochloric acid (m). Percentage germination in media with distilled water is also shown (+). SE bars are shown. Results are from triplicate samples and the experiment was repeated twice. FIG.
siana penetration
of M. sanguinipes cuticle after treatment with 1 mM oxalic, citric, hydrochloric acid, or distilled water. No gross structural differences could be observed in acid-treated cuticle. Most of the B. bassiana penetrating M. sanguinipes cuticle showed the typical penetration peg entering the cuticle at a shallow depression (Figs. 5A-5C). No differences could be observed in the morphology of penetration in the various cuticle treatments. However, in
During the electron microscope scanning of the cuticles, three categories of B. bassiuna were recorded, i.e., (i) conidia which had not germinated, (ii) had germinated but not penetrated the cuticle, and (iii) which had germinated and penetrated the cuticle (Table 3). No differences were observed in the percentage of B. bassiana conidia which had not germinated on cuticle pretreated with distilled water or citric, oxalic, or hydrochloric acids. This verifies the data presented in Figure 4, i.e., germination is
ACIDS
113
independent of acid type. However, the percentage of B. bassiana conidia which had germinated but not penetrated M. sanguinipes cuticle which had been pretreated with hydrochloric acid or distilled water was lower than the citric acid or oxalic acidtreated cuticle. No difference was observed between the hydrochloric acid-treated cuticle or the distilled water-treated cuticle. Conversely, the percentage of B. bassiuna conidia which had germinated and penetrated oxalic acid or citric acid-treated cuticle was higher than the hydrochloric acid or distilled water-treated cuticle. This data indicated that although germination on M. sanguinipes cuticle is not acid-type dependent (also Fig. 4), efficiency of penetration into cuticle is acid-type dependent. DISCUSSION We present the first report of the possible role of metabolic acids produced by any entomopathogenic fungus in virulence against an insect. B. bassiana produced oxalic and citric acids in liquid cultures containing M. sanguinipes cuticle as the sole source of carbon and nitrogen. Furthermore, analysis of the bioassay data showed that the citric acid- or oxalic acid-B. bassiana interaction was synergistic in causing M. sanguinipes mortality. It was previously postulated that oxalic acid produced by entomopathogenic fungi is involved in insect pathogenesis (Charnley, 1984) but to date experimental evidence was lacking. It is clear, however, that in certain phytopathogenic fungi, oxalic acid is important in the destruction of plant tissues and in synergism with cell wall degrading enzymes. Calcium oxalate crystals have been observed on sugar beet leaves infected by the phytopathogenic fungus Sclerotium rolfsii (Punja and Jenkins, 1984). Similarly, oxalic acid-like crystals have been observed on the surface of insects infected by B. bassiana (Dresner, 1950). Oxalic acid alone caused marked localized injury of bean hypocotyls (Bateman and Beer, 1965) and oxalic acid is directly
114
BIDOCHKA
AND KHACHATOURIANS
bassiana interaction with Melanoplus sanguiFIG. 5. Scanning electron micrographs of Beauveria cuticle. Cuticle was pretreated with (A) distilled water, (B) oxalic acid, or (C-F) citric acid. co, conidia; gt. germ tube; ap, appressoria: mu, mucus. Arrows refer to point of penetration of the cuticle. Bar in (A) is 10 pm and applies to (A) through (E). Bar in F is 10 km. nipes
B. bassiuna
TABLE
METABOLIC
3
PERCENTAGE GERMINATION (G) AND PENETRATION (P) OF B. bassiana, AS OBSERVED BY SCANNING ELECTRON MICROSCOPY, ON COMMINUTED M. sanguinipes CUTICLE PRETREATED BY 1 mM ACIDS OR DISTILLED WATER Category”
Cuticle treatment Citric acid Oxalic acid HCl acid Distilled water
-G 47.7
+G,
-P
+G,
+P
(4.6)
20.3 (2.3)
51.3 (1.5)
19.3 (4.7)
53
(2.7)
29.7 (1.3)
32 (61 29.4 (5.8) 17.3 (1.2)
53.7 (2.6)
30.7 (1.2)
15.6 (3.8)
Nore. -G, no germination; +G, -P, germination but no penetration; + G, + P, germination and penetration of the cuticle. N = 500. a Results are expressed as percentage (SE).
toxic to some plants (Franceschi and Horner, 1980). A treatment consisting of S. rolfsii polygalacturonase plus oxalic acid caused extreme degradation of hypocotyl tissue. In a study using 48 isolates of S. rolfsii which infected sugar beet seedlings, Punja et al. (1985) concluded that the determinants of pathogenicity included the ability to produce sufficient quantities of oxalic acid and polygalacturonase and rapid fungal growth. Furthermore, the production of oxalic acid by S. rolfsii in infected tissue and its synergistic interaction with polygalacturonase appeared to function by creating an acid environment favorable for polygalacturonase activity (Bateman and Beer, 1965). To our knowledge, the involvement of citric acid produced by phytopathogenic or entomopathogenic fungi in host infection has never been documented. Citric acid is a primary metabolic product and is formed in the tricarboxylic acid (TCA) cycle by the condensing action of citrate lyase on acetyl-CoA and oxalacetate. Many species of fungi excrete citric acid as a product of primary metabolism and certain fungal strains are used to produce citric acid commercially (Crueger and Crueger, 1984). During the commercial production of citric acid, oxalic acid may be produced as an unwanted byproduct. Oxalic acid production by B. bassiana was found to coincide with an increase in pH to
ACIDS
115
8. It is well known that one of the prerequisites for oxalic acid production in some fungi is a medium pH close to or above neutrality (Kubicek et al., 1988; Crueger and Crueger, 1984; Wilson, 1971). Biosynthetic pathways of oxalic acid or citric acid formation in B. bassiana have not been studied. There is evidence to suggest that in other fungi oxalate is formed through the oxidation of glyoxylate (Balmforth and Thomson, 1984) or the splitting of oxalacetate to form oxalate (Lenz et al., 1976). Both mechanisms involved the role of the TCA or the glyoxylate cycle. However, Kubicek et al. (1988) have shown that in Aspergillus niger oxalate is formed by the action of oxaloacetate hydrolase found in the cytoplasm and does not involve the TCA cycle. The solubilization of insect cuticle proteins by various acids has not been investigated; however, the hydrolysis of noninsect proteins by inorganic and organic acids has been investigated. Our studies did not discern whether the cuticular proteins were actually hydrolyzed or were solubilized. Therefore we chose the term solubilization to describe the release of soluble proteins from the insoluble insect cuticle. There are several reports on the hydrolysis of elastin and other proteins by the action of oxalic, formic, hydrochloric, and acetic acid solutions (Adair et al., 1951; Partridge et al., 1955; Kokoglu, 1978; Partridge and Davis, 1955; Inglis et al., 1980). Hydrolysis of elastin by oxalic acid results in the release of two protein species: a-protein (6& 84 kDa) and P-protein (5.5 kDa; Partridge and Davis, 1955). Oxalic acid hydrolyzes elastin at certain reactive aspartic acid residues (Adair et al., 1951). Generally, the hydrolysis of proteins by acids occurs at an aspartic acid residue whereby the aliphatic carboxyl group of aspartic acid catalyzes the cleavage of both its N- and C-peptide bonds (Inglis et al., 1980). Consistent with this is the fact that aspartic acid, followed by glutamic acid, is released in the free form during elastin hydrolysis (Partridge
116
BIDOCHKA
AND KHACHATOURIANS
and Davis, 1955). Aspartic acid and glutamic acid are the sites of protein hydrolysis by acids. It is not unlikely that M. sanguinipes cuticle is predisposed to acid hydrolysis by the mechanism of aspartic or glutamic acid cleavage since previous analysis has shown the presence of 39.1 and 44.8 aspartic acid and glutamic acid residues, respectively, from 1000 residues of M. sunguinipes cuticular proteins (Bidochka, 1989). Thus, it is possible that M. sanguinipes cuticle is amenable to acid hydrolysis through N- and C-peptide bond cleavage at the aspartic acid or glutamic acid residue. Our analysis revealed no detectable free amino nitrogen after acid hydrolysis of M. sanguinipes cuticle and Inglis et al. (1980) suggested that the Cpeptide bond of aspartic acids is cleaved much more rapidly than the N-peptide of this residue. Thus amino acid liberation by the action of acids is not a criterion for protein hydrolysis. Our results showed that the corresponding oxalate or citrate anions are also involved in M. sunguinipes cuticle solubilization. The mechanism of cuticle solubilization by oxalate and citrate may involve the loosening of the bonds between protein molecules in the cuticle. Our results show that treatment of M. sanguinipes with oxalic acid or citric acid alone caused mortality; treatment of M. sanguinipes with hydrochloric acid did not cause mortality. The reduction in protein bonding in the cuticle by oxalate or citrate may cause the deterioration of cuticle integrity and may lead to the increased permeability and dehydration of the insect. The reduction in protein bonding in the cuticle may also facilitate hyphal penetration by mechanical means or may facilitate the action of extracellular enzymes. The increase of hyphal penetration into oxalic acid- or citric acid-treated cuticle may also reflect an alteration in germ tube orientation toward the cuticle. Another mechanism of action may involve oxalic acid interaction with cuticular calcium. Polygalacturonase produced by the phyto-
pathogenic fungus, S. rofiii, could not hydrolyze calcium pectate, but enzymatic hydrolysis proceeded in the presence of oxalate ions. In plant pathogens, the oxalic acid ties up calcium in the pectates of the bean cell wall thus making the substrate amenable to hydrolysis by polygalacturonase (Bateman and Beer, 1965). We have detected the presence of 427 pg calcium/gm M. sanguinipes cuticle (unpubl. data) and it has been detected in other insect cuticle (Hackman and Goldberg, 1958). Perhaps the oxalic acid produced by B. bussiunu during cuticle penetration ties up the calcium in the cuticle and therein makes the cuticle more amenable to hydrolysis by B. bussiunu protease (Bidochka and Khachatourians, 1987) or chitinases. The production of other metabolites, including metabolic acids, may have a significant role in insect cuticle solubilization or hydrolysis by fungal entomopathogens. ACKNOWLEDGMENTS Research was supported by a grant from the Saskatchewan Agriculture Development Fund. The technical assistance of P. Eckstein is gratefully acknowledged.
REFERENCES W. S. 1925. A method of computing the effectiveness of an insecticide. .I. Econ. Entomol., 18,
ABBOTT,
265-267. G. S., DAVIS, H. F., AND PARTRIDGE, S. M. 1951. A soluble protein derived from elastin. Nuture, 167, 605.
ADAIR,
ANDERSEN,
S. O.,
AND
WEIS-FOGH,
T. 1!264.
Resilin:
A rubberlike protein in arthropod cuticle. In “Advances in Insect Physiology” (J. W. L. Beament, J. H. Treheme, and V. B. Wigglesworth, Eds.), Vol. 2, pp. l-65. Academic Press, New York. ANDERSEN, S. 0. 1980. Cuticular sclerotization. In “Cuticle Techniques in Arthropods” (T. A. Miller, Ed.), pp. 185-215. Springer-Verlag, New York. BALMFORTH, A. J., AND THOMSON, A. 1984. Isolation and characterization of glyoxalate dehydrogenase from the fungus Sclerotium rolfsii. Eiochem. J., 218, 113-118. BATEMAN, D. F., AND BEER, S. V. 1%5. Simultaneous production and synergistic action of oxalic acid and polygalacturonase during pathogenesis by Sclerotium rolfsii. Phytopathology, 55, 204-211. BIDOCHKA, M. J., AND KHACHATOURIANS, G. G.
E. bassiana METABOLIC 1987. Purification and properties of an extracellular protease produced by the entomopathogenic fungus Beauveria
bassiana.
Appl.
Environ.
Microbial.,
53,
1679-1684. BIDOCHKA, M. J. 1989. “Interaction of the Entomopathogenic Fungus Beauveriu bassianu, with the Migratory Grasshopper, Melunoplus sanguinipes: A Systematic Study of Pathogenesis.” Ph.D thesis, University of Saskatchewan. BIDOCHKA, M. J., AND KHACHATOURIANS, G. G. 1990. Identification of Beauveria bassianu extracellular protease as a virulence factor in pathogenicity toward the migratory grasshopper, Melanoplus sanguinipes.
J. Invertebr.
Puthol.,
56, 362-370.
BRADFORD, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal.
Biochem.,
72, 248-254.
CHARNLEY, A. K. 1984. Physiological aspects of destructive pathogenesis in insects by fungi: A speculative review. In “Invertebrate-Microbial Interactions” (J. M. Anderson, A. D. M. Raynor, and D. W. H. Walton, Eds.). pp. 229-270. Cambridge University Press, London. CORDON, T. C., AND SCHWARTZ, J. H. 1962. The fungus Beauveriu tenellu. Science, 138, 1265-1266. CRUEGER, W., AND CRLJEGER,A. 1984. “Biotechnology: A Textbook of Industrial Microbiology” (T. D. Brock, Ed.). Science Tech. Inc., Madison, Wisconsin. DRESNER, E. 1950. The toxic effects of Beauveria bassiana (Bals.) Vuill. on insects. J. N.Y. Entomol. Sot.,
58, 269-278.
EL-SAYED, G. N., COUDRON, T. A., AND IGNOFFO, C. M. 1989. Chitinolytic activity and virulence associated with native and mutant isolates of an entomopathogenic fungus, Nomuraeu rileyi. J. Invertebr.
Pathol.,
54, 39UO3.
FRANCESCHI, V. R., AND HORNER, H. T., JR. 1980. Calcium oxalate crystals in plants. Bot. Rev., 46, 361-427. HACKMAN, R. H., AND GOLDBERG, M. 1958. Proteins of the larval cuticle of Agriunome spinicollis (Coleoptera). J. Insect Physiol., 2, 221-231. GOEIITEL, M. S., ST. LEGER, R. J., RIZZO, N. W., STAPLES, R. C., AND ROBERTS, D. W. 1989. Ultrastructural localization of a cuticle-degrading protease produced by the entomopathogenic fungus Mefarhizium anisopliae during penetration of host (Manduca sexta) cuticle. J. Cert. Microbial., 135, 2233-2239.
INGLIS, A., MCKERN,
N., ROXBURGH, C., AND
117
ACIDS
STRIKE, P. 1980. Cleavage of proteins at aspartic acid residues using dilute acid. In “Methods in Peptide and Protein Sequence Analysis” (C. H. R. Birr, Ed.), pp. 329-343. EIsevier/North-Holland Biochemical Press, Amsterdam. KOKOGLU, E., YENSON, M., AND ERYUREK, F. 1978. Soluble proteins derived from partial mild hydrolysis with oxalic acid of eiastin, fibrin, gluten, and globin and their comparisons by elastolysate test. Med. Bull. Istanbul, 11, 95-99. KUBICEK, C. P., SCHREFERL-KUNAR, G., WOHRER, W., AND ROHR, M. 1988. Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger.
Appl.
Environ.
Microbial.,
54, 633-637.
LENZ, H., WUNDERWALD, P., AND EGGERER, H. 1976. Partial purification and some properties of oxalacetase from Aspergillus niger. Eur. J. Biochem., 65, 225-236.
MARAVALHAS, N. 1970. New technique of protein hydrolysis and analysis of the amino acids on an autoanalyzer. J. Chromatogr., 50, 413-418. PARTRIDGE, S. M.. AND DAVIS, H. F. 1950. Preferential release of aspartic acid during hydrolysis of proteins. Nature, 165, 62-63. PARTRIDGE, S. M., AND DAVIS, H. F. 1955. The chemistry of connective tissues. 3. Composition of soluble proteins derived from elastin. Biochem. J., 61, 21-30.
PARTRIDGE, S. M., DAVIS, H. F., AND ADAIR, G. S. 1955. The chemistry of connective tissues. 2. Soluble proteins derived from the partial hydrolysis of elastin. Biochem. J.. 61, 11-21. PUNJA, Z. K.. AND JENKINS, S. F. 1984. Light and scanning electron microscopic observations of calcium oxalate crystals produced during growth of Sclerotium rolfsii in culture and in infected tissue. Can. J. Bot.,
62, 2028-2032.
PUNJA, Z. K., HUANG, J. S., AND JENKINS, S. F. 1985. Relationship of mycelial growth and production of oxalic acid and cell wall degrading enzymes to virulence in Sclerotium rolfiii. Can. J. Plant Pathol., 7, 109-117. ST. LEGER, R. J., DURRANDS, P. K., CHARNLEY, A. K., AND COOPER, R. M. 1988. Role of extracellular chymoelastase in the virulence of Metarhizium unisopliae for Manduca sexta. J. Invertebr. Puthol.. 52, 285-292.
WILSON, B. J. 1971. Miscellaneous Aspergillus toxins. In “Microbial Toxins” (A. Ciegler and S. Kadis, Eds.). Vol. VI, pp. 207-295. Academic Press, New York.
OF INVERTEBRATE
PATHOLOGY
58,
106-117 (1991)
The Implication of Metabolic Acids Produced by Beauveria in Pathogenesis of the Migratory Grasshopper, Melanoplus MICHAEL Bioinsecticide
J. BIDOCHKA
Research Laboratory, University of Saskatchewan,
bassiana
sanguinipes
AND GEORGE Department Saskatoon,
G. KHACHATOURIANS’
of Applied Microbiology Saskatchewan, Canada
and Food Science, S7N OWO
Received May 23. 1990; accepted September 25, 1990 The entomopathogenic fungus, Beauveria bassiana, produced oxalic and citric acids in liquid cultures containing grasshopper (Mefanoplus sanguinipes) cuticle as the sole nutrient source. Oxalic and citric acids solubilized cuticular proteins as well as commercial preparations of elastin and collagen. Other organic acids which solubilized cuticular proteins included acetic and formic acids. In contrast to these organic acids, the inorganic acids, hydrochloric acid and sulfuric acid, did not solubilize or only slightly solubilized cuticular proteins when compared to the organic acids. M. sanguinipes treated with B. bassiana showed a LT,, of 7.33 days, while M. sanguinipes treated with citric acid or oxalic acid showed a LT,, of 7.25 and 13.28 days, respectively. M. sanguinipes treated with citric acid followed by a B. bassiana conidia treatment showed a LT,, of 3.88 days. and the oxalic acid. B. bassiana treatment resulted in a LT,, of 5.08 days. Analysis of the bioassay data revealed that the relationship between oxalic acid or citric acid together with B. bassiana conidia in grasshopper mortality was markedly synergistic. We suggest that acid metabolites produced by B. bassiana may play a role in cuticle solubihzation and subsequent hyphal penetration. 0 1991 Academic Press. Inc. KEY WORDS: Beauveria bassiana; Melanoplus sanguinipes: migratory grasshopper; metabolic acids; oxalic acid; citric acid: pathogenesis.
INTRODUCTiON
The initial stages of insect infection by entomopathogenic fungi include the penetration of the host cuticle. Several studies have shown that penetration of the cuticle is accomplished by the action of fungal extracellular enzymes accompanied by mechanical pressure of the advancing hyphae. For example, extracellular protease produced by Beauveria bassiana (Bidochka and Khachatourians, 1987, 1990) and Metarhizium anisopliae (St. Leger et al., 1988) are important determinants of fungal virulence toward the migratory grasshopper, Melanoplus sanguinipes, and the tobacco hornworm. Manduca sexta, respectively. Chitinase produced by Nomuraea rileyi is an important enzyme in pathogenicity toward Trichoplusia ni (El-Sayed et al., 1989). Mechanical pressure is also associated with M. anisopliae penetration of M. ’ To whom reprint requests should be addressed.
sexta procuticle
(Goettel et al., 1989). Other factors such as fungal metabolic acids which may contribute in the solubilization or hydrolysis of insect cuticle have only been speculated upon (Charnley, 1984). but experimental evidence is lacking. Many species of fungi produce an assortment of metabolites, including various organic acids. Of the entomopathogenic fungi, Beauveria tenella produces oxalic acid (Cordon and Schwarz, 1962) and oxalic acid-like crystals have been observed on the surface of insects infected by B. bassiana (Dresner, 1950). Oxalic acid solubilizes elastin (Kokoglu et al., 1978; Partridge et al., 1955; Adair et al., 1951) and elastinlike (i.e., resilin) tissue comprises part of the cuticle of certain insects (Andersen and Weis-Fogh, 1964). For analytic purposes, dilute acids, including oxalic acid, have been used to partially hydrolyze proteins into smaller peptides for further determination of the amino acid composition (Maravalhas, 1970). In fact, the use of dilute 106
0022-201 l/91 $1.50 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
B. bassiana
METABOLIC
acids is one of the oldest and simplest chemical methods for fragmenting protein chains (Partridge and Davis, 1950). In the present study we confirm the production of oxalic acid by B. bassiana and present the first report on the production of citric acid by this fungus in liquid cultures containing insect cuticle as the sole source of nutrients. Citric acid was studied since oxalic acid is a by-product of citric acid formation in fungi (Crueger and Crueger, 1984). The effects of these metabolic acids and other acids on the solubilization of cutitular proteins of the migratory grasshopper, M. sanguinipes, are reported. Finally, the possible synergistic role of metabolic acids during pathogenesis of B. bassiana is examined by pretreating M. sanguinipes with dilute acid solutions and then conducting a bioassay using B. bassiana. This is the first such study which investigates the possible role of metabolic acids produced by this entomopathogenic fungus in the insect infection process. MATERIALS
AND METHODS
Preparation of B. bassiana conidia and rearing conditions of the migratory grasshopper, M. sanguinipes, were described previously (Bidochka and Khachatourians, 1990). The insoluble proteins, elastinorcein and analytical grade elastin, were prepared from bovine neck ligament, and collagen was prepared from bovine achilles tendon (Sigma Chemical Co., St. Louis, Missouri). The acids which were used were of reagent grade and were diluted with sterile-distilled water to yield 0.1 mM to 1 M solutions. Preparation of ground M. sanguinipes cuticle. The method of Andersen (1980) for the bulk preparation of M. sanguinipes cuticle was used. Frozen grasshoppers, which had previously been starved for 24 hr, were homogenized in a Waring blender containing 1% (w/v) potassium tetraborate (100 grasshoppers, of equal sex ratio, per liter solution). The material was collected from
ACIDS
107
a plastic sieve (l-mm screen), dried, and milled to a tine powder (
Ox-
alic acid production and citric acid production by B. bassiana grown in liquid cultures were determined by enzymatic analysis using Boehringer Mannheim test kits (Federal Republic of Germany). Insoluble proteins and grasshopper cuticle treatment with acids. The ability of var-
108
BIDOCHKA
AND
ious acids to solubilize the following insoluble proteins was tested: elastin-orcein, analytical grade elastin, collagen, or the protein components of M. sanguinipes powdered cuticle. The structural proteins, collagen and elastin, are insoluble in distilled water. However, peptide components of these proteins may be solubilized if the intact protein is treated with certain acids. Ten milligrams of one of these insoluble proteins or M. sanguinipes powdered cuticle was incubated in 2 ml of 0.1 mM to 1 M organic acid (oxalic, citric, boric, or formic acid) solutions. Likewise ammonium and sodium salts of oxalic and citric acids were also tested. The suspensions were incubated at 27°C for 24 hr, then passed through membrane filters (HA-type, 0.45-kmpore-size). Solubilization of elastin-orcein was measured as the release of a chromophore which absorbs at 520 nm. Solubilization of analytic grade elastin, collagen, or powdered M. sanguinipes cuticle was measured as the release of soluble protein and free amino nitrogen.
KHACHATOURIANS
dipped in acid were not washed subsequent to the B. bassiana treatment. The 3-set acid dip is sufficient to wet the grasshoppers, they then appeared dry after 1 hr. One group was dipped into the conidial suspension only; this is the B. bassiana treatment. Of the control groups, one group was dipped in sterile-distilled water and one group was left untreated. The grasshoppers were individually housed and placed in an environmental chamber (27°C 30% relative humidity) and fed barley grass daily at which time the mortality was checked. Probit analysis was performed on Abbott corrected data (Abbott, 1925). Analysis of synergism between citric acid or oxalic acid and B. bassiana during the bioassay against M. sanguinipes was done using the “Dose-Effect Analysis with Microcomputers” software program (Elsevier Science Publ., The Netherlands) operated on an Apple Be computer. Scanning electron microscopy. Pieces of M. sanguinipes cuticle (approx 0.5 cm2) were excised from the abdomen. Carefully, Soluble protein and free amino nitrogen any loose tissue was scraped away without determination. Soluble protein was deterdamaging the cuticle. The cuticle was mined by the protein dye-binding assay of washed with sterile-distilled water and a Bradford (1976) using bovine y-globulin as streptomycin solution (100 pg/ml), then inwater, 1 mM hythe standard. Free amino nitrogen was de- cubated in sterile-distilled termined by the trinitrobenzene sulfonic drochloric acid, citric acid, or oxalic acid acid assay using glycine as the standard, for 1 hr. The cuticle was then placed on a and this assay was previously described by sterile Whatman No. 2 filter paper which Bidochka and Khachatourians (1990). had been soaked in water and placed in a Bioassay procedures. Adult M. sanguisterile Petri dish. Ten microliters of lo5 conipes were incapacitated with carbon dioxnidia/ml (approx lo3 conidia) was placed on ide. Each of the seven treatment groups the surface of the cuticle. The Petri dish and the two control groups consisted of 60 was sealed with parafilm and placed in an grasshoppers. Three groups of grasshopenvironmentally controlled chamber for 48 pers were dipped, for 3 set, into a 1 mM hr at 27°C. The cuticle pieces were then hydrochloric, oxalic, or citric acid solution, fixed in 2.5% glutaraldehyde in 10 mM porespectively. These are the acid treatments. tassium phosphate buffer (PH 7) for 24 hr, Three groups were dipped into one of the dehydrated in a series of ethanol solutions three acid solutions, then after 1 hr, each and then 100% acetone. The samples were grasshopper was dipped into a B. bassiana critical point dried in a Polaron drier and conidial suspension (10’ conidia/ml) for 3 then coated with gold in a Technics sec. These are the acid + B. bassiana treat“Hummer” sputter coater. Upper surfaces ments. The grasshoppers which were of the cuticle were viewed and photo-
B. bassiana
METABOLIC
graphed through a Cambridge Instrument Stereoscan Mark 2A scanning electron microscope operated at -20 kV. RESULTS Production of Metabolic Acids by B. bassiana Grown in Liquid Culture Containing Grasshopper Cuticle
Figure 1 shows the production of citric and oxalic acids by B. bassiana during growth on a 1% (w/v) suspension of M. sanguinipes powdered cuticle. Fifty percent of the conidia germinated within 24 hr. Oxalic acid production was low at day 2 (2.8 pg/ml) but increased at a rate of 50 pg oxalic acid/ml/day from days 2 to 3. Thereafter, oxalic acid levels remained at approximately 100 pg/ml. Similarly, citric acid showed three phases of production: (1) a low level of production up to day 3 at a rate of 7 kg/ml/day, (2) followed by an increase in the level of production from days 3 to 5 at a rate of 26 p.g/ml/day, and (3) and a decrease in citric acid levels up to day 6. Expressed in terms of molar equivalents at the highest levels of production, oxalic acid and citric acid levels were approximately 0.8 and 0.3 mM, respectively. Although the acid production increased with culture age the culture pH increased. This suggests that
8.5 8 7.5 I
-
2
0 Culture
4 Age
zl
6 (days)
1. Production of citric acid (0) and oxalic acid by Beauveria bassiana in liquid culture containing 1% (w/v) Melanoplus sanguinipes powdered cuticle. The pH (A) of the culture is also shown. Assays were performed in triplicate from duplicate cultures. SE bars are shown. FIG.
(0)
ACIDS
109
metabolic bases, possibly ammonia, were also being produced in cultures. Oxalic and citric acids were also formed in liquid media containing various combinations of 1% (w/v> gelatin, 1% (w/v) glucose, and 0.1 M CaCl,. There was no increase in oxalic acid or citric acid production in media containing CaCl,. We also checked if these acids originated from insect cuticle. Ten milligrams of cuticle was washed with 2 ml of 10 mM HCI in order to solubilize any calcium oxalate. We did not detect the presence of oxalic acid at levels of sensitivity of the assay (2 Fg/ml). Solubilization of Elastin and Grasshopper Cuticle by Various Acid
Table 1 shows the release of soluble products from elastin-orcein, elastin, collagen, and M. sanguinipes cuticle by various inorganic and organic acids or by distilled water alone. When compared with other acids, citric acid and oxalic acid showed the highest amount of elastin-orcein solubilization. Incubation of elastin-orcein in 1 M boric acid or distilled water resulted in the lowest solubilization of elastin-orcein. Since the soluble chromophore could be detected in the distilled water treatment, we suspect that a certain amount of watersoluble chromophore is nonspecifically released. No proteins were solubilized in water if the substrate was elastin or collagen. A small amount of protein was solubilized from elastin or collagen by the inorganic acids when compared with the organic acids. Of the organic acids, incubation of elastin with citric acid showed the highest release of soluble protein, and formic acid had the greatest activity on collagen. When the M. sanguinipes cuticle is suspended in distilled water, 2.6% of the dry weight of the cuticle was solubilized as protein. Since approximately 61% of the dry weight of M. sanguinipes cuticle is protein (Bidochka, 1989), the amount solubilized by distilled water represents about 4.2% of the total cutitular proteins by weight. Citric acid had the highest effect on solubilization of M.
110
SOLUBILIZATION
BIDOCHKA
OF ELASTIN-ORCEIN.
AND
KHACHATOURIANS
TABLE 1 ELASTIN, COLLAGEN. AND Melanoplus CONCENTRATIONS OF VARIOUS ACIDS
sanguinipes
CUTICLE
BY 1 M
Substrate’ Treatment Inorganic acid Hydrochloric Sulfuric Organic acid Acetic Boric Citric Formic Oxalic Water
Elastin-orcein
Elastin
Collagen
0.06 (0.01) 0.04 (0.02)
0.07 (0.01) 0
0.17 (0.03) 0.07 (0.02)
0.06 0.03 0.07 0.04 0.07
1.89 1.09 2.48 1.81 1.11
2.65 0.88 2.94 3.94 1.29
(0.01) (0.01) (0.01) (0.01) (0.02)
0.03 (0.02)
0
(0.17) (0.13) (0.07) (0.27) (0.07)
0
(0.24) (0.03) (0.10) (0.03) (0.20)
Cuticle
1.04 (0.18) 1.43 (0.37) 8.78 1.24 12.22 10.32 2.97
(1.87) (0.83) (0.66) (2.22) (0.69)
2.58 (0.24)
a Solubilization of elastin-orcein measured by release of a soluble chromophore detected at A,,,. Solubilization of elastin, collagen, and powdered M. sanguinipes cuticle was measured as the release of protein and expressed as the percentage (*SD) of the total dry weight from triplicate samples. The experiment was repeated twice with similar results.
cuticle. Approximately 12% of the dry weight of cuticle was solubilized as protein and this represents about 20% of the total cuticular proteins. Hydrochloric acid had the lowest amount of soluble protein release from cuticle. Of the organic acids, boric acid showed the lowest release of soluble protein from M. sanguinipes cuticle. The liberation of free amino nitrogen could not be detected in any of the samples in the range of sensitivity of the trinitrobenzene sulfonic acid assay. We were primarily interested in the effects of B. bassiana acid metabolites, especially oxalic and citric acids, on M. sanguinipes cuticle solubilization. We next examined the stoichiometric relationship between concentrations of oxalic acid and citric acid solutions and M. sanguinipes cuticle solubilization. Furthermore, we compared these effects to those of the salt forms of these acids, namely ammonium oxalate and sodium citrate, on cuticle solubilization. Figure 2A shows the release of soluble protein from M. sanguziupes cuticle by 0 to 1 M concentrations of oxalic acid or ammonium oxalate. The release of soluble proteins by the action of oxalic acid alone was highest at a 0.01 M concentration (Fig. 2A). sanguinipes
The release of soluble protein from M. sanguinipes cuticle decreased with oxalic acid concentrations greater or less than 0.1 M. It is interesting to note that the release of soluble protein by 1 M oxalic acid is only slightly higher than the effect of distilled water alone. This confirms the data in Table 1 which show that the release of soluble protein from M. sanguinipes cuticle by the action of 1 M oxalic acid solubilizes approximately 0.5% more protein when compared to the distilled water control treatment. On the other hand, the release of soluble protein from M. sanguinipes cuticle by the action of 0.01 M oxalic acid solubilizes approximately 5.5% more protein when compared to the distilled water control treatment. Figure 2B shows the release of soluble protein from M. sanguinipes cuticle by 0 to 1 M concentrations of citric acid or sodium citrate. Here the shape of the curve is quite different than that of the action of oxalic acid on the release of soluble protein from M. sanguinipes cuticle. The effect of citric acid on M. sanguinipes cuticle shows a saturation kinetics curve; a rapid increase of soluble protein from 0 to 0.1 M and a saturation of soluble protein release with citric
B. bassiana METABOLIC
ACIDS
111
values of 13.28 and 7.25 days. Hydrochloric acid treatment had no effect on M. sanguinipes when compared with the untreated controls. M. sanguinipes pretreated with hydrochloric acid followed by a B. bassi0.000 0.025 0.050 ana conidia treatment did not significantly reduce the LT,, values when compared to the B. bassiana conidia alone treatment. M. sanguinipes treated with oxalic or citric o!..,..r.‘,..n”,.‘, acid followed by a treatment with B. bassi0.0 0.2 0.4 0.6 0.8 1.0 1.2 ana conidia had lower LT,, values than the Oxalate [Ml singular treatments. There were no differences in M. sanguinipes mortality between the group which was dipped in steriledistilled water and the untreated group. The possibility of a synergistic effect between oxalic or citric acid and B. bassiana conidia on M. sanguinipes mortality was evaluated using a “Dose-Effect Analysis 0.025 0.050 with Microcomputers” software. Figure 3 00 shows the results of these analyses. The 0.0 0.2 0.4 0.6 0.8 1.0 1.2 combination index refers to the antagonisCitrate [Ml tic or synergistic interaction between two FIG. 2. Solubilization of Melanoplus sanguinipes treatments. A combination index greater cuticular proteins. Ten milligrams of powdered M. than 1 indicates that an interaction is antagsanguinipes cuticle was incubated in 2 ml of (A) oxalic acid (Cl) or ammonium oxalate (H) and (B) citric acid onistic; less than 1, the interaction is syn(0) or sodium citrate (W) solutions for 24 hr and sol- ergistic and an additive effect is indicated uble protein was measured. Insets show detailed reby a combination index equal to 1. The sults of protein solubilization for 0 to 0.05 M concenanalysis of the relationship between citric trations. Results are from triplicate assays. SE was acid and B. bassiana indicated that there less than 10% of the mean for each datum point. The experiment was repeated twice. was marked antagonism between citric acid and B. bassiana at low effect levels (i.e., ~20% of M. sanguinipes affected) but synacid concentrations greater than 0.1 M. No ergism at higher effect levels. The analysis free amino acids were detected as a result of the relationship between oxalic acid and of the action of citric or oxalic acid on M. B. bassiana indicated that there was slight sanguinipes cuticle. antagonism between citric acid and B. bassiana at effect levels less than 1% of M. Interactive Effects of Oxalic or Citric sanguinipes affected and markedly synerAcid and B. bassiana Pathogenesis gistic at higher effect levels. Therefore. at a low mortality of M. sanguinipes there is anTable 2 shows the summary for the probit tagonism between the oxalic or citric acids analyses for M. sanguinipes (i) treated with and B. bassiana. Another way the data may B. bassiana conidia, (ii) treated with 1mM oxalic, citric, or hydrochloric acids, and be interpreted is that 80% of the mortality (iii) pretreated with acid followed by a B. of M. sanguinipes in the B. bassiana-citric bassiana conidia treatment. The LTso for acid bioassay may be explained by the synM. sanguinipes treated with B. bassiana ergistic relationship between the two comof M. conidia was 7.33 days. Citric and oxalic ac- ponents. Likewise, 99% mortality ids also killed M. sanguinipes with LTso sanguinipes in the B. bassiana-xalic acid
112
BIDOCHKA
AND KHACHATOURIANS TABLE
PROBIT
ANALYSIS
DATA
FOR THE Beauveria
2
bassiana/AcrD-Melanoplus
sanguinipes
SYSTEM
Regression line equationd
LT,, fiducial limits’ Treatment”
Lower
LT,n
Upper
Intercept
Slope
conidia only Oxalic only Citric only HCl only* Oxalic + B. bassiana conidia Citric + B. bassiana conidia HCl + B. bassiana conidia
6.91 10.56 6.62 4.66 3.67 6.80
7.33 13.28 7.25 5.08 3.88 7.17
7.78 16.65 8.52 5.53 4.09 7.54
-1.54 0.29 1.21 -0.61 -3.67 -5.67
3.51 2.22 3.04 3.29 5.46 6.08
B. bassiana
a Oxalic, oxalic acid; citric, citric acid; HCl, hydrochloric acid. b LT,, values could not be calculated for the HCl-treated M. sanguinipes since no mortality differences could be observed when compared with the controls. ’ Fiducial limits and times in days (P = 0.05). d Equation of the best-fitting line using the maximum-likelihood method. Sample size for each treatment was 60 grasshoppers and the experiment was repeated once with similar results.
bioassay may be explained by the synergistic relationship between the two components. On the whole, the analyses indicated a marked synergistic action between oxalic acid or citric acid with B. bassiana. Effect of pH and the Associated Bases of Oxalic, Citric, or Hydrochloric Acid on B. bassiana Germination
The above date show that one of the possible factors responsible for the synergism between oxalic and citric acid and B. bassiana is the role of acids in solubilizing M. sanguinipes cuticle (Table 1, Fig. 2). Oxalic
citric
acid
oxalic
0 0.0 Fraction
"I.'I'.I"I..l 0.2
acid
0.4 of
t
LM
Bb t Bb
0.6 sanguinipes
0.8
1.0 affected
FIG. 3. The analysis of the interaction of citric acid plus Beauveria bassiana (Bb) or oxalic acid plus Bb in a bioassay against Melanoplus sanguinipes. The combination index refers to antagonism (>l), additive effects (= 1). or synergism (Cl).
or citric acid may loosen the cuticular proteins to facilitate hyphal penetration. Another possible explanation for the synergism between oxalic and citric acid and B. bassiana is the role of pH or the associated base on the germination B. bassiana conidia. The organic acid may lower the pH and the associated base may act as a nutrient source to facilitate conidial germination. To test this hypothesis, B. bassiana conidia were incubated in a 1% (w/v) M. sanguinipes cuticle medium with varying pHs adjusted with 1, 10, or 100 mM oxalic, citric, or hydrochloric acid and conidial germination was counted after a 24-hr incubation. Hydrochloric acid was included as the control inorganic acid. Figure 4 shows the results of this experiment. No differences in percentage of B. bassiana conidia germination were observed in the M. sanguinipes cuticle suspensions adjusted to approximately similar pH and similar molarity. Therefore, conidial germination is pH dependent but not dependent on the corresponding bases of oxalic, citric, or hydrochloric acid. SEM of Acid-Treated Grasshopper Cuticle and B. bassiana Penetration
Figure 5 shows some examples of B. bas-
B. bassiuna Molarity of 0.01 --0.1
o! 1
METABOLIC
Acids 0.001 -
I
I.
I
I.
I
,
2
3
4
5
6
7
PH
4. The percentage of Beauveriu bassiuna conidia germinating after 24 hr in liquid medium containing 1% (w/v) Melunoplus sanguinipes cuticle with 0.1, 0.01, or 0.001 M oxalic acid (O), citric acid (0), or hydrochloric acid (m). Percentage germination in media with distilled water is also shown (+). SE bars are shown. Results are from triplicate samples and the experiment was repeated twice. FIG.
siana penetration
of M. sanguinipes cuticle after treatment with 1 mM oxalic, citric, hydrochloric acid, or distilled water. No gross structural differences could be observed in acid-treated cuticle. Most of the B. bassiana penetrating M. sanguinipes cuticle showed the typical penetration peg entering the cuticle at a shallow depression (Figs. 5A-5C). No differences could be observed in the morphology of penetration in the various cuticle treatments. However, in
During the electron microscope scanning of the cuticles, three categories of B. bassiuna were recorded, i.e., (i) conidia which had not germinated, (ii) had germinated but not penetrated the cuticle, and (iii) which had germinated and penetrated the cuticle (Table 3). No differences were observed in the percentage of B. bassiana conidia which had not germinated on cuticle pretreated with distilled water or citric, oxalic, or hydrochloric acids. This verifies the data presented in Figure 4, i.e., germination is
ACIDS
113
independent of acid type. However, the percentage of B. bassiana conidia which had germinated but not penetrated M. sanguinipes cuticle which had been pretreated with hydrochloric acid or distilled water was lower than the citric acid or oxalic acidtreated cuticle. No difference was observed between the hydrochloric acid-treated cuticle or the distilled water-treated cuticle. Conversely, the percentage of B. bassiuna conidia which had germinated and penetrated oxalic acid or citric acid-treated cuticle was higher than the hydrochloric acid or distilled water-treated cuticle. This data indicated that although germination on M. sanguinipes cuticle is not acid-type dependent (also Fig. 4), efficiency of penetration into cuticle is acid-type dependent. DISCUSSION We present the first report of the possible role of metabolic acids produced by any entomopathogenic fungus in virulence against an insect. B. bassiana produced oxalic and citric acids in liquid cultures containing M. sanguinipes cuticle as the sole source of carbon and nitrogen. Furthermore, analysis of the bioassay data showed that the citric acid- or oxalic acid-B. bassiana interaction was synergistic in causing M. sanguinipes mortality. It was previously postulated that oxalic acid produced by entomopathogenic fungi is involved in insect pathogenesis (Charnley, 1984) but to date experimental evidence was lacking. It is clear, however, that in certain phytopathogenic fungi, oxalic acid is important in the destruction of plant tissues and in synergism with cell wall degrading enzymes. Calcium oxalate crystals have been observed on sugar beet leaves infected by the phytopathogenic fungus Sclerotium rolfsii (Punja and Jenkins, 1984). Similarly, oxalic acid-like crystals have been observed on the surface of insects infected by B. bassiana (Dresner, 1950). Oxalic acid alone caused marked localized injury of bean hypocotyls (Bateman and Beer, 1965) and oxalic acid is directly
114
BIDOCHKA
AND KHACHATOURIANS
bassiana interaction with Melanoplus sanguiFIG. 5. Scanning electron micrographs of Beauveria cuticle. Cuticle was pretreated with (A) distilled water, (B) oxalic acid, or (C-F) citric acid. co, conidia; gt. germ tube; ap, appressoria: mu, mucus. Arrows refer to point of penetration of the cuticle. Bar in (A) is 10 pm and applies to (A) through (E). Bar in F is 10 km. nipes
B. bassiuna
TABLE
METABOLIC
3
PERCENTAGE GERMINATION (G) AND PENETRATION (P) OF B. bassiana, AS OBSERVED BY SCANNING ELECTRON MICROSCOPY, ON COMMINUTED M. sanguinipes CUTICLE PRETREATED BY 1 mM ACIDS OR DISTILLED WATER Category”
Cuticle treatment Citric acid Oxalic acid HCl acid Distilled water
-G 47.7
+G,
-P
+G,
+P
(4.6)
20.3 (2.3)
51.3 (1.5)
19.3 (4.7)
53
(2.7)
29.7 (1.3)
32 (61 29.4 (5.8) 17.3 (1.2)
53.7 (2.6)
30.7 (1.2)
15.6 (3.8)
Nore. -G, no germination; +G, -P, germination but no penetration; + G, + P, germination and penetration of the cuticle. N = 500. a Results are expressed as percentage (SE).
toxic to some plants (Franceschi and Horner, 1980). A treatment consisting of S. rolfsii polygalacturonase plus oxalic acid caused extreme degradation of hypocotyl tissue. In a study using 48 isolates of S. rolfsii which infected sugar beet seedlings, Punja et al. (1985) concluded that the determinants of pathogenicity included the ability to produce sufficient quantities of oxalic acid and polygalacturonase and rapid fungal growth. Furthermore, the production of oxalic acid by S. rolfsii in infected tissue and its synergistic interaction with polygalacturonase appeared to function by creating an acid environment favorable for polygalacturonase activity (Bateman and Beer, 1965). To our knowledge, the involvement of citric acid produced by phytopathogenic or entomopathogenic fungi in host infection has never been documented. Citric acid is a primary metabolic product and is formed in the tricarboxylic acid (TCA) cycle by the condensing action of citrate lyase on acetyl-CoA and oxalacetate. Many species of fungi excrete citric acid as a product of primary metabolism and certain fungal strains are used to produce citric acid commercially (Crueger and Crueger, 1984). During the commercial production of citric acid, oxalic acid may be produced as an unwanted byproduct. Oxalic acid production by B. bassiana was found to coincide with an increase in pH to
ACIDS
115
8. It is well known that one of the prerequisites for oxalic acid production in some fungi is a medium pH close to or above neutrality (Kubicek et al., 1988; Crueger and Crueger, 1984; Wilson, 1971). Biosynthetic pathways of oxalic acid or citric acid formation in B. bassiana have not been studied. There is evidence to suggest that in other fungi oxalate is formed through the oxidation of glyoxylate (Balmforth and Thomson, 1984) or the splitting of oxalacetate to form oxalate (Lenz et al., 1976). Both mechanisms involved the role of the TCA or the glyoxylate cycle. However, Kubicek et al. (1988) have shown that in Aspergillus niger oxalate is formed by the action of oxaloacetate hydrolase found in the cytoplasm and does not involve the TCA cycle. The solubilization of insect cuticle proteins by various acids has not been investigated; however, the hydrolysis of noninsect proteins by inorganic and organic acids has been investigated. Our studies did not discern whether the cuticular proteins were actually hydrolyzed or were solubilized. Therefore we chose the term solubilization to describe the release of soluble proteins from the insoluble insect cuticle. There are several reports on the hydrolysis of elastin and other proteins by the action of oxalic, formic, hydrochloric, and acetic acid solutions (Adair et al., 1951; Partridge et al., 1955; Kokoglu, 1978; Partridge and Davis, 1955; Inglis et al., 1980). Hydrolysis of elastin by oxalic acid results in the release of two protein species: a-protein (6& 84 kDa) and P-protein (5.5 kDa; Partridge and Davis, 1955). Oxalic acid hydrolyzes elastin at certain reactive aspartic acid residues (Adair et al., 1951). Generally, the hydrolysis of proteins by acids occurs at an aspartic acid residue whereby the aliphatic carboxyl group of aspartic acid catalyzes the cleavage of both its N- and C-peptide bonds (Inglis et al., 1980). Consistent with this is the fact that aspartic acid, followed by glutamic acid, is released in the free form during elastin hydrolysis (Partridge
116
BIDOCHKA
AND KHACHATOURIANS
and Davis, 1955). Aspartic acid and glutamic acid are the sites of protein hydrolysis by acids. It is not unlikely that M. sanguinipes cuticle is predisposed to acid hydrolysis by the mechanism of aspartic or glutamic acid cleavage since previous analysis has shown the presence of 39.1 and 44.8 aspartic acid and glutamic acid residues, respectively, from 1000 residues of M. sunguinipes cuticular proteins (Bidochka, 1989). Thus, it is possible that M. sanguinipes cuticle is amenable to acid hydrolysis through N- and C-peptide bond cleavage at the aspartic acid or glutamic acid residue. Our analysis revealed no detectable free amino nitrogen after acid hydrolysis of M. sanguinipes cuticle and Inglis et al. (1980) suggested that the Cpeptide bond of aspartic acids is cleaved much more rapidly than the N-peptide of this residue. Thus amino acid liberation by the action of acids is not a criterion for protein hydrolysis. Our results showed that the corresponding oxalate or citrate anions are also involved in M. sunguinipes cuticle solubilization. The mechanism of cuticle solubilization by oxalate and citrate may involve the loosening of the bonds between protein molecules in the cuticle. Our results show that treatment of M. sanguinipes with oxalic acid or citric acid alone caused mortality; treatment of M. sanguinipes with hydrochloric acid did not cause mortality. The reduction in protein bonding in the cuticle by oxalate or citrate may cause the deterioration of cuticle integrity and may lead to the increased permeability and dehydration of the insect. The reduction in protein bonding in the cuticle may also facilitate hyphal penetration by mechanical means or may facilitate the action of extracellular enzymes. The increase of hyphal penetration into oxalic acid- or citric acid-treated cuticle may also reflect an alteration in germ tube orientation toward the cuticle. Another mechanism of action may involve oxalic acid interaction with cuticular calcium. Polygalacturonase produced by the phyto-
pathogenic fungus, S. rofiii, could not hydrolyze calcium pectate, but enzymatic hydrolysis proceeded in the presence of oxalate ions. In plant pathogens, the oxalic acid ties up calcium in the pectates of the bean cell wall thus making the substrate amenable to hydrolysis by polygalacturonase (Bateman and Beer, 1965). We have detected the presence of 427 pg calcium/gm M. sanguinipes cuticle (unpubl. data) and it has been detected in other insect cuticle (Hackman and Goldberg, 1958). Perhaps the oxalic acid produced by B. bussiunu during cuticle penetration ties up the calcium in the cuticle and therein makes the cuticle more amenable to hydrolysis by B. bussiunu protease (Bidochka and Khachatourians, 1987) or chitinases. The production of other metabolites, including metabolic acids, may have a significant role in insect cuticle solubilization or hydrolysis by fungal entomopathogens. ACKNOWLEDGMENTS Research was supported by a grant from the Saskatchewan Agriculture Development Fund. The technical assistance of P. Eckstein is gratefully acknowledged.
REFERENCES W. S. 1925. A method of computing the effectiveness of an insecticide. .I. Econ. Entomol., 18,
ABBOTT,
265-267. G. S., DAVIS, H. F., AND PARTRIDGE, S. M. 1951. A soluble protein derived from elastin. Nuture, 167, 605.
ADAIR,
ANDERSEN,
S. O.,
AND
WEIS-FOGH,
T. 1!264.
Resilin:
A rubberlike protein in arthropod cuticle. In “Advances in Insect Physiology” (J. W. L. Beament, J. H. Treheme, and V. B. Wigglesworth, Eds.), Vol. 2, pp. l-65. Academic Press, New York. ANDERSEN, S. 0. 1980. Cuticular sclerotization. In “Cuticle Techniques in Arthropods” (T. A. Miller, Ed.), pp. 185-215. Springer-Verlag, New York. BALMFORTH, A. J., AND THOMSON, A. 1984. Isolation and characterization of glyoxalate dehydrogenase from the fungus Sclerotium rolfsii. Eiochem. J., 218, 113-118. BATEMAN, D. F., AND BEER, S. V. 1%5. Simultaneous production and synergistic action of oxalic acid and polygalacturonase during pathogenesis by Sclerotium rolfsii. Phytopathology, 55, 204-211. BIDOCHKA, M. J., AND KHACHATOURIANS, G. G.
E. bassiana METABOLIC 1987. Purification and properties of an extracellular protease produced by the entomopathogenic fungus Beauveria
bassiana.
Appl.
Environ.
Microbial.,
53,
1679-1684. BIDOCHKA, M. J. 1989. “Interaction of the Entomopathogenic Fungus Beauveriu bassianu, with the Migratory Grasshopper, Melunoplus sanguinipes: A Systematic Study of Pathogenesis.” Ph.D thesis, University of Saskatchewan. BIDOCHKA, M. J., AND KHACHATOURIANS, G. G. 1990. Identification of Beauveria bassianu extracellular protease as a virulence factor in pathogenicity toward the migratory grasshopper, Melanoplus sanguinipes.
J. Invertebr.
Puthol.,
56, 362-370.
BRADFORD, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal.
Biochem.,
72, 248-254.
CHARNLEY, A. K. 1984. Physiological aspects of destructive pathogenesis in insects by fungi: A speculative review. In “Invertebrate-Microbial Interactions” (J. M. Anderson, A. D. M. Raynor, and D. W. H. Walton, Eds.). pp. 229-270. Cambridge University Press, London. CORDON, T. C., AND SCHWARTZ, J. H. 1962. The fungus Beauveriu tenellu. Science, 138, 1265-1266. CRUEGER, W., AND CRLJEGER,A. 1984. “Biotechnology: A Textbook of Industrial Microbiology” (T. D. Brock, Ed.). Science Tech. Inc., Madison, Wisconsin. DRESNER, E. 1950. The toxic effects of Beauveria bassiana (Bals.) Vuill. on insects. J. N.Y. Entomol. Sot.,
58, 269-278.
EL-SAYED, G. N., COUDRON, T. A., AND IGNOFFO, C. M. 1989. Chitinolytic activity and virulence associated with native and mutant isolates of an entomopathogenic fungus, Nomuraeu rileyi. J. Invertebr.
Pathol.,
54, 39UO3.
FRANCESCHI, V. R., AND HORNER, H. T., JR. 1980. Calcium oxalate crystals in plants. Bot. Rev., 46, 361-427. HACKMAN, R. H., AND GOLDBERG, M. 1958. Proteins of the larval cuticle of Agriunome spinicollis (Coleoptera). J. Insect Physiol., 2, 221-231. GOEIITEL, M. S., ST. LEGER, R. J., RIZZO, N. W., STAPLES, R. C., AND ROBERTS, D. W. 1989. Ultrastructural localization of a cuticle-degrading protease produced by the entomopathogenic fungus Mefarhizium anisopliae during penetration of host (Manduca sexta) cuticle. J. Cert. Microbial., 135, 2233-2239.
INGLIS, A., MCKERN,
N., ROXBURGH, C., AND
117
ACIDS
STRIKE, P. 1980. Cleavage of proteins at aspartic acid residues using dilute acid. In “Methods in Peptide and Protein Sequence Analysis” (C. H. R. Birr, Ed.), pp. 329-343. EIsevier/North-Holland Biochemical Press, Amsterdam. KOKOGLU, E., YENSON, M., AND ERYUREK, F. 1978. Soluble proteins derived from partial mild hydrolysis with oxalic acid of eiastin, fibrin, gluten, and globin and their comparisons by elastolysate test. Med. Bull. Istanbul, 11, 95-99. KUBICEK, C. P., SCHREFERL-KUNAR, G., WOHRER, W., AND ROHR, M. 1988. Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger.
Appl.
Environ.
Microbial.,
54, 633-637.
LENZ, H., WUNDERWALD, P., AND EGGERER, H. 1976. Partial purification and some properties of oxalacetase from Aspergillus niger. Eur. J. Biochem., 65, 225-236.
MARAVALHAS, N. 1970. New technique of protein hydrolysis and analysis of the amino acids on an autoanalyzer. J. Chromatogr., 50, 413-418. PARTRIDGE, S. M.. AND DAVIS, H. F. 1950. Preferential release of aspartic acid during hydrolysis of proteins. Nature, 165, 62-63. PARTRIDGE, S. M., AND DAVIS, H. F. 1955. The chemistry of connective tissues. 3. Composition of soluble proteins derived from elastin. Biochem. J., 61, 21-30.
PARTRIDGE, S. M., DAVIS, H. F., AND ADAIR, G. S. 1955. The chemistry of connective tissues. 2. Soluble proteins derived from the partial hydrolysis of elastin. Biochem. J.. 61, 11-21. PUNJA, Z. K.. AND JENKINS, S. F. 1984. Light and scanning electron microscopic observations of calcium oxalate crystals produced during growth of Sclerotium rolfsii in culture and in infected tissue. Can. J. Bot.,
62, 2028-2032.
PUNJA, Z. K., HUANG, J. S., AND JENKINS, S. F. 1985. Relationship of mycelial growth and production of oxalic acid and cell wall degrading enzymes to virulence in Sclerotium rolfiii. Can. J. Plant Pathol., 7, 109-117. ST. LEGER, R. J., DURRANDS, P. K., CHARNLEY, A. K., AND COOPER, R. M. 1988. Role of extracellular chymoelastase in the virulence of Metarhizium unisopliae for Manduca sexta. J. Invertebr. Puthol.. 52, 285-292.
WILSON, B. J. 1971. Miscellaneous Aspergillus toxins. In “Microbial Toxins” (A. Ciegler and S. Kadis, Eds.). Vol. VI, pp. 207-295. Academic Press, New York.