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Journal of Venomous Animals and Toxins

Print version ISSN 0104-7930On-line version ISSN 1678-4936

J. Venom. Anim. Toxins vol.5 n.2 Botucatu  1999 

Original paper



PURIFICATION AND CHARACTERIZATION OF TOXINS FROM WHEAT ISOLATES OF Drechslera tritici-repentis, Bipolaris bicolor, AND Bipolaris sorokiniana



1 Center of Biotechnology, Instituto Biológico, 2 Department of Phytopathology, Escola Superior de Agricultura "Luiz de Queiroz", University of São Paulo – USP, State of São Paulo, Brazil.



ABSTRACT. Low molecular weight metabolites produced by Bipolaris bicolor, Bipolaris sorokiniana, and Drechslera tritici-repentis are considered to be toxins that facilitate disease in wheat cultivars. Several such toxins were isolated from these fungi. Electrophoresis demonstrated bands of proteins that reduced shoot inhibition in susceptible plants but not in resistant plants. Chlorophyll content was reduced during the first 10 hours of light in the susceptible plants and after 18 hours in the resistant plants. The enzyme beta-1,3-glucanase increased in the resistant plants after treatment with toxins, but in the susceptible plants this enzyme decreased compared to the control. This suggests that the toxin was a protein and the susceptible plants needed other mechanisms for induced resistance.
 KEY WORDS: toxin, wheat pathogens, protein toxin.




Wheat diseases including leaf spots and root rots caused by Bipolaris bicolor, Bipolaris sorokiniana, and Drechslera tritici-repentis are an important cause in the reduction of grain productivity. The presence of toxic metabolites in fungal culture filtrates of wheat-Pyrenophora tritici-repentis have been reported in several studies (4,8,20,21), but only in one for wheat-Bipolaris (12).

Pyrenophora tritici-repentis produces low-molecular weight toxins in vitro (20). Ballance et al. (3) demonstrated that culture filtrates of certain Pyrenophora tritici-repentis isolates also contained low molecular weight (MW 13,900 and 14,700) host-selective, non-dialyzable, and heat sensitive toxins. Therefore, unlike most fungal phytotoxins, the toxin from Pyrenophora tritici-repentis had a high molecular weight and was probably a protein (13). Tomas et al. (21) also describe the purification of toxin from Pyrenophora tritici-repentis, observing a protein. For Bipolaris spp. there are no studies available.

In the this study, we investigated the involvement of a toxic compound obtained from the isolates of Bipolaris bicolor, Bipolaris sorokiniana, and Drechslera tritici-repentis in the mechanism of resistance and effect on chlorophyll concentration in wheat plants.



PATHOGEN CULTURE. Four isolates of Bipolaris bicolor, ten of Bipolaris sorokiniana, and four of Drechslera tritici repentis were obtained from infected wheat seeds or leaves collected from the field and then maintained on potato-dextrose-agar (PDA) plates. One hundred milliliter of modified Fries medium (20) were incubated with mycelial plugs of the fungus grown on PDA for 10 days. Bottles were incubated without agitation for 21 days at 25ºC with constant light.

TOXIN PATHOGEN ISOLATION. Mycelium was removed by vacuum filtration with Whatman No.1 to remove the remaining hyphae. The culture filtrates were concentrated under partial vacuum at 40ºC to one fifth of the original volume. Equal volumes of cold (4ºC) methanol were then added. The mixture was refrigerated overnight and any subsequent precipitate discarded. Methanol was removed from the water phase by rotary evaporation and vacuum at 40ºC, and an equal volume of chloroform was added. The water phase was separated from the chloroform, and an equal volume of ether was added. The ether was removed and the water phase was evaporated under vacuum at 40ºC for the remaining ether. The water phase was used for bioassays. The treatment of the controls the same procedure was used with modified non-inoculated growth medium. The extracts were submitted to protein (11) and phenol (19) quantification.

TOXIN BIOASSAYS. Wheat seeds of cultivars IAC-24 (susceptible) and BH-1146 (resistant) were germinated in Petri dishes (size 1.2 cm) with sheets of moist filter paper for four days at 25ºC and 12 h of light. Germinated seeds of 0.5cm in height were placed in beakers (10 ml) containing 2 ml of toxin solutions at concentrations of 3 mg, 1.5 mg, and 0.5 mg of serum albumin bovine (SAB)/ml. For the controls, 2 ml of culture medium and 2 ml of water were used. Those plants were maintained at 25ºC and constant light. Two days later, the length of the aerial plants and root were measured, and the aspect and color of the root were observed.

Detached leaves of the susceptible cultivar (IAC-24) and the resistant cultivar (BH-1146) were nicked with a needle and the damaged leaf surfaces were immediately covered with 20 µl of toxins. The leaves were incubated at 24ºC in a moist chamber and the nicked area was observed 48 h after inoculation for the development of spot or necrosis.

EXTRACTION AND CHLOROPHYLL CONTENT. One gram of leaf tissue was triturated with five milliliters of a solution acetone/water (80:20), filtered through cheesecloth, and the volume adjusted to 10 ml. The samples ware transferred to a cuvette and the OD value at 652 nm was read in a Pye Unicam spectrophotometer against acetone blank. The chlorophyll content was calculated using Arnon's equation (1), and its decrease was estimated using the controls (wheat plants treated with water or medium).

In another phase, the chlorophyll content in leaf extracts treated with toxin at a concentration of 3 mg of SAB/ml was analyzed at 0 to 50-hour intervals. These were made for five isolates representing B. bicolor (2 strains), B. sorokiniana (2 strains), and D. tritici-repentis (1 strain). The result was a correlation between time and inhibition level. After this procedure, chlorophyll was extracted from the treated wheat cultivars with all isolates at a concentration of 3 mg of SAB/ml after 32 hours of observation.

ELECTROLYTE LEAKAGE. The leaves were washed with water, cut into small slices (1cm in length and 5mm in width), and transferred to a beaker containing 1 ml of water. Conductances of ambient solutions were measured at 0 to 48-hour intervals. Conductance in µmho of control leaves treated with water was subtracted from that of the toxin-treated leaves to determine any increase or not in electrolyte leakage. The same method was used for root slices. A series of assays were run with triplicate vials.

EXTRACTION OF SOLUBLE PROTEINS. Two grams of toxin-treated wheat leaves and controls (water and medium) were triturated in ice with 10 ml acetate buffer, pH 5, 0.05M. After 30 minutes at 4ºC, the extracts were filtered through Whatman No.1, centrifuged at 10.000 g for 10 min, and the activity of enzyme beta-1,3-glucanase was analyzed following methods described by Van Hoof (22) and Lever (10). The extracts were also submitted to protein (11) and phenol (19) quantification.

GEL ELECTROPHORESIS. Sodium dodecyl sulfate gel electrophoresis (horizontal slab, LKB) was performed in 7% acrylamide gels using 0.1M Tris-glycine buffer system, pH 8.2, with 2% SDS. Gels were stained for proteins in 50% methanol, 12% acetic acid, 0.5% trichloroacetic acid, 0.1% Coomassie Brilliant Blue R-250, and destained in 20% methanol and 12% acetic acid.

SAMPLE PREPARATION FOR GEL-ELECTROPHORESIS. A lyophilized protein sample (400 mg) on equal protein basis was dissolved with 1 ml of prepared sample buffer. The sample buffer solution contained 100 ml 0.1M Tris-glycine, pH 8.2, and 2 g of SDS. This sample was heated in water-bath at 100ºC for 5 min, cooled at room temperature, and 22 µl of gel per slot was added.

ASSAY FOR TOXIC ACTIVITY OF GEL-ELECTROPHORESIS BANDS. Samples from 20 slots were removed from gel-electrophoresis bands and homogenized with 1 ml of 0.1M Tris-glycine, pH 8.2. Germinated seeds (0.5 cm in height) were placed in beakers (10 ml) containing 1 ml of electrophoresis solutions and compared with controls in beakers containing 1 ml of Tris-glycine 0.1M, pH 8.2. Those plants were maintained at 25ºC and constant light. Two days later, the length of the aerial plants and root were measured.



QUANTIFICATION OF PROTEINS AND PHENOLS FROM TOXINS. All toxins prepared from wheat isolates (B. bicolor, B. sorokiniana and Drechslera tritici-repentis) showed a protein concentration level between 3 and 6 mg of SAB/ml and phenols between 0.4 and 0.5 mg of chlorogenic acid/ml.

EFFECT OF TOXIN ON THE SHOOT INHIBITION BIOASSAY. Toxins prepared in three dilutions with sterile distilled water induced seedling shoot inhibition, and significant differences were observed between the resistant and susceptible cultivars (Table 1). Shoot inhibition decreases as dilution increases, however, at the concentration of 0.5 mg of SAB/ml, we observed no effect on the resistant seedlings. The plants treated with water and medium did not show differences. The susceptible plants treated with toxins were brown in color and the tissues were brittle. This enhanced color was due to the high toxic concentration. The susceptible plants treated with water or medium showed white roots.

INOCULATION IN DETACHED LEAVES. All toxins obtained from isolates at concentrations of 3 to 1.5 mg of SAB/ml induced necrosis in leaves from both wheat cultivars one to two days after inoculation. For toxins from B. bicolor and B. sorokiniana at a concentration of 0.5mg of SAB/ml necrosis was not observed in leaves from resistant cultivars, but in leaves from the susceptible plants small spots of necrosis were observed. For Drechslera tritici-repentis toxins at a concentration of 0.5 mg of SAB/ml necrosis was observed three to four days after inoculation in both cultivars. All the treatments were compared with results obtained from the controls (water and medium).

CHLOROPHYLL CONTENT IN SUSCEPTIBLE AND RESISTANT WHEAT LEAVES. A reduction in chlorophyll content was observed in wheat leaves treated with toxins at 0 to 50 hour-intervals. The reduction in chlorophyll of susceptible wheat leaves began after 10 h of treatment for all toxins. However, after 24 hours toxins from Drechslera tritici-repentis and Bipolaris sorokiniana showed a greater inhibition than that of toxins from Bipolaris bicolor. For the resistant plants, the reduction in chlorophyll began after 18 hours of treatment with toxins. However, toxins from Drechslera tritici-repentis showed a higher inhibition in chlorophyll content after 32 hours (Figure 1). The dose/time response data (32 h/3 mg/SAB), which showed chlorophyll inhibition, were different between the wheat cultivars when the effect with all toxins obtained from B. bicolor, B. sorokiniana, and Drechslera tritici-repentis was analyzed. For the resistant wheat cultivars, the toxins from Drechslera tritici-repentis showed 40% inhibition in chlorophyll content, while toxins from B. bicolor and B. sorokiniana showed only 10% inhibition. However, the susceptible wheat plants showed 30% inhibition for B. bicolor toxins, 40-50% inhibition for B. sorokiniana toxins, and 60-70% inhibition for Drechslera tritici-repentis toxins (Figure 2).


FIGURE 1. Effects of the toxins from Bipolaris bicolor (Bb), B. sorokiniana (Bs), and Drechslera tritici-repentis (Dtr) on cholorophyll content (mean percentage inhibition in relation to controls) at 0-50-hour intervals in IAC-24 and BH-1146 wheat varieties.




FIGURE 2. Effects of the toxins from B. bicolor (Bp), B. sorokiniana (Bs), and Drechslera tritici-repentis (Dtr) at a concentration of 3mg SAB/ml 32 hours after treatment on chlorophyll content (mean percentage inhibition in relation to controls), in IAC-24 and BH-1146 wheat varieties.


ELECTROLYTE LEAKAGE. The experiment using leaf tissues or root treated with toxins did not show a greater leakage of electrolyte than the control leaves exposed to water up until 50 hours. Therefore, inhibition of chlorophyll synthesis at 32 hours or more is not associated with leaky membranes.

QUANTIFICATION OF BETA-1,3-GLUCANASE. The activity of beta-1,3-glucanase was markedly increased in the resistant wheat plants treated with all toxins when compared with those treated with water and medium. In contrast, the susceptible wheat plants treated with all toxins did not show any increase of beta-1,3-glucanase in relation to the controls. A characterization of the enzyme was then performed to determine induction during the treatment with toxin (Figure 3).


FIGURE 3. Beta-1,3-glucanase activity in leaves of resistant (BH-1146) and susceptible (IAC-24) wheat plants treated with toxins from isolates Bipolaris bicolor (Bb), Bipolaris sorokiniana (Bs), and Drechslera tritici-repentis (Dtr).



ELECTROPHORETIC ANALYSES OF TOXINS. SDS-PAGE of toxins showed one peak with molecular weights of approximately 14.0 kDa for B. bicolor, 9.0 kDa for B. sorokiniana, and 18.0 kDa for Drechslera tritici-repentis (Figure 4).


FIGURE 4. SDS-PAGE of toxins from B. bicolor, B. sorokiniana, and Drechslera tritici-repentis with molecular weights (standard).



ASSAY FOR TOXIC ACTIVITY OF GEL-ELECTROPHORESIS BANDS. Samples of 20 slots from gel-electrophoresis in contact with germinated seeds induced seedling shoot inhibition, and significant differences were observed between the resistant and susceptible cultivars (Table 2).


TABLE 2. Effects of the toxins from Bipolaris bicolor (Bb), B. sorokiniana (Bs) and Drechslera tritici-repentis (Dtr) extracted by gel electrophoresis on the shoot inhibition assay in the IAC-24 and BH-1146 wheat cultivars.




Toxin can be defined as a compound produced by a microbial pathogen that causes damage to the host plant and is involved in the development of plant disease (16). The compounds usually included in this category are low molecular weight, which disrupt the highly integrated physiological process of the host, giving rise to symptoms such as wilting, chlorosis, and necrosis (17,18).

The reaction of wheat cultivars to Bipolaris bicolor, Bipolaris sorokiniana, and Drechslera tritici-repentis was evaluated with a rating scale in lesion type/concentration of conidia suspension and resistant cultivars (BH-1146) were then identified. Susceptibility to isolates was characterized by the presence of tan necrosis, and resistance by the absence of symptoms (2).

Lamarie & Bernier (8), Hosford et al. (5), and Tomas & Bockus (20) studied the pathogen Pyrenophora tritici-repentis, which produced toxic compounds when grown in culture. These toxins appear to be active only against wheat species and are classified as toxin host-selective. In this study, we produced toxins in culture for Drechslera tritici-repentis, Bipolaris bicolor, and Bipolaris sorokiniana. A bioassay was performed to analyze the toxic effect in shoot inhibition and also to observe the reaction in detached leaves after toxin inoculation. Toxicity activity was found in all isolates when analyzed in wheat cultivars at a concentration of 3 mg of SAB/ml of toxin. As toxins are diluted with sterile nutrient medium or water, their effects decrease and at 0.5mg of SAB/ml the resistant plants did not show root inhibition. Detached leaves were divided into two categories, such as susceptible and resistant leaves. The susceptible leaves showed spots with necrotic lesions, while the resistant leaves exhibited slight spots.

To identify differences in toxigenicity, Ballance et al. (3) reported that Pyrenophora tritici-repentis isolates might produce high-molecular weight toxins in vitro (estimated in 13,900) with chlorosis and necrosis, which are called Ptr-necrosis-toxins. Brown & Hunger (4) reported that Ptr-toxin has a low molecular weight (800 to 1,800) and is related to the production of chlorosis. Orolaza et al. (14) reported that Ptr-necrosis-toxin elicited extensive chlorosis in susceptible wheat plants and appeared to be a pathogenicity factor. In this work, we have studied not only the effect on lesion or shoot development inhibition but also the correlation of the toxin effect with chlorophyll and its biochemical mechanisms.

The toxin at the concentration of 3mg SAB/ml induced root development inhibition in both cultivars, and the chlorophyll content was analyzed at 0 to 50 hour-intervals. It is important to note that the susceptible plants show a reduction in the chlorophyll in the first 10 hours of light, while in the resistant plants this reduction began in the first 18 hours. This indicated that resistant plants have a protection and this was prolonged for another 8 hours after the beginning of inhibition in the susceptible plants. Also after 32 hours of treatment, the toxin from Drechslera tritici-repentis showed 40% inhibition for the resistant plants and 60% inhibition for the susceptible plants. The chlorophyll content was reduced, but no damage was observed in the cell because there was no electrolytic leakage.

Isaac (6) reported that fungi produce a wide range of toxic compounds with different biochemical structures and modes of action, including polypeptides, glycoproteins, amino acid derivatives, etc. The mechanisms of toxicity of those compounds, which have been satisfactorily associated with disease development, are still largely unknown, but can have more than one chemically related toxic component. This suggests a variety of molecular mechanisms for toxicity and specificity. Microscopic observations (9) have shown the penetration of Pyrenophora tritici-repentis into wheat leaves through resistant and susceptible cultivars and suggested that a molecular mechanism might be influencing their resistance. In the susceptible tissue, however, the continued growth of the parasite might allow the production of toxic substances to the host, so that lesions occur rapidly. Based on these observations, growth in the resistant cultivar never progresses, perhaps due to previous molecular mechanisms. Many proteins or groups of proteins are induced to respond to pathogen attack. The induced synthesis of some of these proteins is thought to result from the activation of host defenses and was called pathogenesis-related (PR) proteins (15). One of these proteins was beta-1,3-glucanase, which was measured in this study.

The enzymatic activity of the susceptible plants was found to be lower than that of the resistant and control plants. This suggests that enzyme can be responsible for defense and can be induced by the toxin.

Electrophoretic analyses indicated bands of protein present in all toxins with molecular weights of approximately 14.0 kDa for Bipolaris bicolor, 9.0 kDa for Bipolaris sorokiniana, and 18.0 kDa for Drechslera tritici-repentis. Shoot inhibition was found to be higher in the susceptible leaves than in resistant leaves by the proteins isolated from gel.

In summary, we purified a toxin structurally characterized as a protein, and this opens an exciting possibility for the study of host-pathogen interactions. Many chemicals are known to induce resistance against fungi (7). Our studies provide evidence that the toxin induces the PR-proteins in the resistant plants but not in the susceptible plants. Our results suggest that the susceptible plants need other mechanisms for induced resistance.



The authors are grateful Dr.Antonio Luiz Gonçalves, in memoriam, for his assistance in revising the manuscript.



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Received 11 August 1997
Accepted 16 February 1998

E. E. BACH – Centro de Biotecnologia, Instituto Biológico, Caixa Postal 12898, CEP 04010-970, São Paulo, Brasil.

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