Introduction

The consumption of wheat in Brazil is more than 10.6 million tons/year, but in the last few years the country did not produce a sufficient amount of wheat to supply its internal demand, having to import about 70% of the wheat consumed [5]. This low production is due to several factors, such as the reduction of tilled areas, the climate conditions with warm and humid winters, which contribute to the development of fungal diseases. [22].

Bipolaris sorokiniana (Sacc. in Sorok) Shoemaker, 1959; (teleomorph): Cochliobolus sativus (Ito & Kuribayashi) Drechsl. Ex Dastur, is a phytopathogenic fungus found worldwide, that causes diseases in wheat and other winter cereals. The diseases caused by this fungus are spot blotch disease, common root rot, and black point of the grain [21]. This fungus has high morphological, physiological, and genetic variability, which makes it difficult to identify and also to determine the appropriate measures for disease control [18, 24–26].

Different methods have been used to study the variability of B. sorokiniana [15, 16, 18, 26]. The presence of enzymes with capacity to degrade the plants cell wall is a method that has been used extensively in the characterization of phytopathogenic fungi variability [7, 9, 20]. Combined with this method, the mycelial growth and virulence in plants provides important information about the pathogen, and can be related to the enzyme activity. Analysis of the protein profile by electrophoresis is a simple and practical method, which has been used in the identification of fungal species and in studies of genetic variability [1, 2, 14]. The main objective of the present work was to study physiological variability and virulence of B. sorokiniana monoconidial isolates from different geographic regions in Brazil and from other countries, through the analysis of mycelial morphology, enzymatic activity, total protein profile, and virulence.

Materials and Methods

Fungal Isolates

Thirty-five B. sorokiniana isolates were used in this study, being 34 obtained from wheat seeds and one from barley seeds. The Brazilian isolates were supplied by CNTP (EMBRAPA, Brazil), and the others were supplied by CIMMYT (Mexico), as shown in Table 1. Monoconidial cultures were prepared by isolating single conidia from each isolate and growing them on potato dextrose agar (PDA) medium.

Table 1 Bipolaris sorokiniana isolates used in this work, their geographical origin, morphological groups, test, and extracellular enzyme activity determined by H/C mean diameter
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Morphological Groups

Morphological groupings were made based on colonial characteristics and mycelial growth [11]. For this purpose, a 0.5 cm disc with mycelium of each isolate was inoculated in the center of Petri dish with PDA medium and incubated at 25°C with a 12 h photoperiod for 5 days. The morphological analysis consisted of observation of the mycelial color and texture, the formation or not of sectors and the mycelial growth type.

Virulence in Wheat Seeds and Seedlings

The virulence of the isolates was evaluated using wheat seeds of the BRS Buriti cultivar, which are classified as moderately susceptible to B. sorokiniana infection. For inoculum production, mycelial discs of each isolate were transferred to Petri dishes with PDA medium and incubated at 25°C for 7 days. Seeds were inoculated following the method of Tanaka and Menten [23], modified for this experiment. For each isolate, 200 seeds were used, divided in 4 groups of 50 seeds for each fungal isolate [4]. Each seed group was kept for 24 h at 25°C in the Petri dishes with PDA medium colonized with the fungal isolate to be tested. After 24 h, the seeds were removed from the plates and sown in plastic trays. The substratum used was the Plantmax® type, sterilized and irrigated before the sowing. The sown trays were kept in a greenhouse at 25°C for 10 days. The evaluation was carried through determining the number of emerged seedlings (flag leaf with the double of the seed size), healthy or not, with injuries on the leaves (spot blotch), seeds (black point) and colon. The obtained data were analyzed by one-way ANOVA with a level of significance of 0.01 using Microsoft Excel 2007 software.

Extracellular Enzyme Activity

The B. sorokiniana isolates were evaluated for their capacity to produce extracellular enzymes in solid medium. Mycelial discs with 0.5 cm of diameter of each isolate was transferred individually to three Petri dishes with minimum medium (6 g NaNO3, 0.5 g KCl, 1.5 g KH2PO4, 0.5 g MgSO4, 0.01 g ZnSO4, 0.01 g FeSO4, 15 g agar, 1 l distilled H2O) supplied with the enzyme substratum as follow: soluble starch 1% for amylase, carboxymethylcellulose 1% for cellulase, gelatin 4% for protease, citric pectin 1% for pectinase, CaCl2 0.01%, and Tween 80 1% for esterase [8] with pH adjusted to 6.0. Plates were incubated at 25°C for 5 days and then the activity was observed, after the addition of a revealing solution (lugol for amylase, Congo-red for cellulase, (NH4)2SO4 for protease, Cetab for pectinase) or incubation at 4°C for 48 h for esterase.

The enzymatic activity was quantified by the ratio of the mean halo diameter (H) divided by the mean colony diameter (C) in three repetitions. The results were analyzed using the one-way ANOVA using Microsoft Excel 2007 software, with a level of significance of 0.01, followed by the Tukey test with α = 0.05, when necessary.

Total Protein Extraction and Analysis in Polyacrylamide Gel

Ten milligrams of fungal mycelium was collected from the PDA medium. The mycelial mass was ground to a fine powder under liquid nitrogen and homogenized in 0.2 ml of cold extraction buffer (1% SDS, 9 M Urea, 25 mM Tris-HCl pH 6.8, 1 mM EDTA, 0.7 M β-mercaptoethanol). The homogenate was transferred into 1.5-ml Eppendorf tubes, boiled for 3 min, centrifuged for 20 min at 20,000g, and the supernatant was collected. The crude protein extract was used in a 15% polyacrylamide gel SDS-PAGE and the protein separation was done at 150 V for 3 h. After electrophoresis, the gel was stained with 1% Coomassie Blue solution, fixed, dried, and photographed.

Results

Morphological Groups

The 35 B. sorokiniana isolates were divided into five morphological groups on the basis of mycelial morphological variability and growth pattern. These groups were: (I) black fluffy growth with white sectors; (II) black fluffy growth; (III) gray cottony growth; (IV) white cottony growth; and (V) white suppressed growth (Fig. 1). Groups I and III were predominant with 38.88% and 36.11% of the isolates, respectively. Less frequent were groups VI, II, and V with 11.11%, 8.33% and 5.55% of the isolates, respectively (Table 1).

Fig. 1
figure 1

Isolates representing the five morphological groups in PDA medium: a Group I; b Group II; c Group III; d Group IV; and e Group V. 254 × 190 mm (600 × 600 DPI)

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Virulence in Wheat Seeds and Seedlings

The virulence of B. sorokiniana isolates was determined from the percentage of total emerged seedlings, and the symptoms type (Table 2). The symptoms were classified as spot blotch disease in leaves and colon, and typical black point symptoms in seeds. The majority of the roots showed little or no infection (data not shown); on the other hand, almost all seeds with no germination presented symptoms of black point disease. Isolates 98010, 98003, 98030, and 98012 showed the highest degree of virulence, and CS1110 and 98006 showed the lowest, without seedling symptoms.

Table 2 Percentage of virulence of B. sorokiniana isolates in wheat seeds and seedlings, the morphological groups and cluster formation considering these data
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A dendrogram was constructed, using the software SPSS v.17.0, with the data obtained for virulence together with the morphological groups (Fig. 2). The clusters formed with the data showed four groups considering a cutoff of 87% proximity. Group A was formed by the isolates from all morphological groups except for one isolate, CS1004, from group III, which presented a very low virulence on seed. Group B was formed by the isolates with high infectivity (0–33.3% seed germination), represented by morphological groups I and IV. Group C was formed by isolates from morphological groups I and II, with moderate infectivity. Group D was formed only by isolates from the morphological pattern Group III, and three sub clusters were formed: one formed only by isolates with high infectivity, another with moderate infectivity, and one with the isolates which low infectivity.

Fig. 2
figure 2

Dendrogram constructed with the data obtained from the virulence experiment in wheat seeds and seedlings and morphological groups. 190 × 275 mm (600 × 600 DPI)

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The ANOVA test was carried out to assess if different morphological groups differ statistically with respect to emerged seeds and the results showed that the observed difference in emergence of seeds was not statistically significant (F = 1.49).

Extracellular Enzyme Activity

All isolates showed amylase and cellulase activity (Table 1). The highest amylase activity index (H/C = 1.35) was observed for isolate BS18M2 and the lowest was shown by isolate CS1110 (H/C = 0.42). For cellulase activity, isolate 98029 showed the highest index (H/C = 1.36) and 19/92 the lowest (H/C = 0.83). Protease activity was observed for 27 isolates, with the highest index for CS110 (H/C = 1.04). Twenty-two isolates were able to produce pectinase. Of these, isolates 98025 and 98040 showed the highest activities (H/C = 1.36 and H/C = 1.21, respectively). Isolate 98017 showed, among all enzymes tested, the highest index activity (2.95) for esterase (Table 1). The ANOVA test showed that the differences observed in the average enzyme activity for each isolate were not statistically significant (F = 0.69).

The mean H/C ratios of all the isolates in each test ranged from 0.89 for protease to 1.34 for esterase, considering only the isolates with positive results. The ANOVA showed that the differences were statistically significant (F = 18.22). The Tukey test showed that the means of H/C index for esterase activity were significantly different from all enzymes, while the other enzymes did not differ among them.

SDS-PAGE Polyacrylamide Gel Electrophoresis

The SDS-PAGE polyacrylamide gels were analyzed considering the number, size and intensity of the protein bands, being considered only the strong intensities and reproducible bands. Protein profile of the B. sorokiniana isolates showed polymorphism, demonstrating the genetic diversity among them, with variation in the number and intensity of the bands. However, differences in protein profile were observed between isolates from the same morphological group and also from the same geographical region. Isolates from Brazil showed some common bands in the protein profile, indicating a possible intraspecific relationship. Two bands between 43 and 38 kDa were common among 90% of the Brazilian isolates. These profiles were not present in isolates from other countries.

Discussion

The phytopathogenic fungus B. sorokiniana has a high morpho-pathological variability, which has not so far been confirmed at the molecular level [17, 18]. Our results showed a high morphological variability. Five morphological groups were formed, with no relationship to the geographical regions. Nascimento and Van Der Sand [16] did not observe a correlation between genetic similarity groups and geographical origin in B. sorokiniana, inferring that hardly the morphologic characteristics expression would be unique conditional for genes. Thus, this variability could be attributed to the interactions between genetic information, where isolates from the same species differ from one another in their genetic composition, and environmental conditions, such as the edaphoclimatic differences in the areas where the isolates were obtained. On fungal populations, the diversity is the norm, while uniformity is the exception [13].

The results obtained for virulence with the 35 isolates showed that all morphological groups included isolates with high, moderate, and low infectivity. Isolates CS1110 and 98006 had white mycelia, and they did not produce reproductive structures in the culture medium. These isolates would be important, and could be used in the induction of systemic resistance, therefore can colonize the plant without causing diseases, but by activating the plant defense systems they restrain the establishment of the pathogen. Also they may compete with the pathogen for this ecological niche [3].

Oliveira et al. [18] found differences in pathogenicity among the B. sorokiniana isolates; however, this could not be correlated to the morphological characteristics. Jaiswall et al. [11] found a correlation between the virulence and the colonial morphology, in which isolates with black mycelia were most virulent. Similarly, Pandey et al. [19] studied the variability of B. sorokiniana, and were able to correlate morphological variability with virulence. Arabi and Jawhar [2] found three different pathotypes of Cochliobolus sativus, one of which was extremely virulent. Farias et al. [6] carried out an evaluation of the fungi that cause oat helminthosporiosis. Of six species, B. sorokiniana was the most aggressive. Independent of the fact that B. sorokiniana has predominantly asexual, haploid and heterocaryotic reproduction, the variability in virulence suggests that extensive genetic exchange occurs in this species [6]. This may be explained by the presence of nuclei in the mycelium and conidia. The variation can be generated when the exchange of nuclei is followed by nuclear fusion, somatic recombination, and the consequent chromosomal rearrangement for the haploidization.

The H/C enzymatic index equal or greater than 2 is recommended to assure the microorganism ability to degrade a solid medium substrate [12]. Considering this value, only isolates 98017, CS1110, and 98011 got the index higher than 2 for esterase activity, although the ANOVA showed no statistical significance. The isolates tested showed a higher enzymatic activity for esterase. This may be explained by the importance of these enzymes for the initial phase of adherence and invasion of plant tissue by phytopathogenic fungi [10].

The majority of the isolates from Brazil showed different total protein patterns from those from abroad. However, it was not possible to establish a correlation between the total protein pattern and characteristics such as virulence, morphology, and enzymatic activity. A hypothesis for this is that hardly these characteristics are conditional for the same alleles. Consequently, the total protein analysis suggested that this method could be used in genetic diversity studies and in identification of B. sorokiniana.