In vitro effect of Bacillus thuringiensis strains and Cry proteins in phytopathogenic fungi of paddy rice-field

Efeito in vitro de cepas e proteínas Cry de Bacillus thuringiensis em fungos fitopatogênicos da cultura do arroz irrigado

ENVIRONMENTAL MICROBIOLOGY

In vitro effect of Bacillus thuringiensis strains and Cry proteins in phytopathogenic fungi of paddy rice-field

Efeito in vitro de cepas e proteínas Cry de Bacillus thuringiensis em fungos fitopatogênicos da cultura do arroz irrigado

Neiva Knaak^I; Angelise Ana Rohr^I; Lidia Mariana Fiuza^I,II,*

^IUniversidade do Vale do Rio dos Sinos, Laboratório de Microbiologia, São Leopoldo, RS, Brasil

^IIInstituto Riograndense do Arroz Integrado, Cachoeirinha, RS, Brasil

ABSTRACT

Cry1Ab and Cry1Ac strains and proteins synthesized by Bacillus thuringiensis thuringiensis and B. thuringiensis kurstaki were assessed in the following phytopathogens: Rhizoctonia solani,Pyricularia grisea,Fusarium oxysporum and F. solani, which had their micelial growth decreased after incubation in the presence of the bacterial strains. As to Cry proteins, there were no inhibition halo development in the assessed concentrations.

Key-words: Paddy rice; Bacillus thuringiensis; phytopathogens; Cry proteins.

RESUMO

As cepas e proteínas Cry1Ab e Cry1Ac sintetizadas por Bacillus thuringiensis thuringiensis e B. thuringiensis kurstaki, foram avaliadas nos fitopatógenos: Rhizoctonia solani,Pyricularia grisea,Fusarium oxysporum e F. solani, os quais tiveram seu crescimento micelial reduzido após a incubação na presença das cepas bacterianas. Em relação às proteínas Cry, não houve formação de halo de inibição nas concentrações avaliadas.

Palavras-chave: Arroz irrigado, Bacillus thuringiensis, fitopatógenos, proteínas Cry.

Rice is extensively cultivated around the world and it is the nutritional foundation in many countries, including Brazil. The South Brazilian region has its share with 68% of the national production, and Rio Grande do Sul stands out with 46.7% of grown hectares (4). Rice there is first in grain production, followed by barley, oat, and wheat, (13). But all the available technology in nowadays agriculture can not prevent large losses due to diseases and pests. Their control is always determinant for rice culture sustainability (5).

The various diseases which attack paddy rice fields can yield losses and damage that render culture productivity unsteady, and reach about 10 to 15% of the potential production. Among such diseases brusone stands out; it is caused by the Pyricularia grisea fungus (Cooke), of which the damage can yield a production loss between 70 and 80%, and bring forth leaf stains, stalk, panicle and grains. Blade burning is another important disease by the Rhizoctonia solani (Riker e Gooch) fungus. It can cause the death of the bottom leaves, of which the blades are strongly attacked thus leading to the sterilization of some spikelets (5). Other fungi, such as, Fusarium oxysporum (Link e Gray, 1821) which brings forth the badly of the lap, and Fusarium solani (Link e Gray, 1821), both commonly seen in irrigated and upland rice can be found from the very beginning of panicle emission up to the maturational phase, causing great damage for grain and seed quality (27). Thus, it is crucial to find new practices that make it possible the control of these diseases, seeing that the biological control means a viable alternative.

Bacillus genus bacteria has great potential as biological control agents because they keep their viability when stored for long time (1). B. thuringiensis is a Gram-positive bacteria that brings forth crystal inclusions during sporulation, made up of Cry proteins (19,33). They are highly toxic and specific and that is why they are harmless for most non-target organisms (18,30). These toxins are codified by cry genes, and their toxicity has a relationship with the C-terminal section of polypeptidical chains, while the N-terminal section is determinant for crystal structure shape (23). Some B. thuringiensis strains show only one codifying gene (Bt kurstaki HD-73), while other ones show different genes (Bt aizawai 7.29) (22,29).

Hence, this paper wants to assess Cry1Ab and Cry1Ac strains and proteins synthesized by B. thuringiensis thuringiensis 407 (pH 408) and B. thuringiensis kurstaki HD-73, respectively, regarding 4 phytopathogenic fungus strains associated with paddy rice.

Strains of Pyricularia grisea,Rhizoctonia solani,Fusarium oxysporum and F. solani phytopathogenic fungi were provided by the Molecular- Phytopathology Laboratory of Universidade Federal do Rio Grande do Sul, and they were growing in medium plates (Potato dextrose agar- PDA) and kept at 4ºC.

B. thuringiensis thuringiensis 407 (pH 408) and B. thuringiensis kurstaki HD-73 strains were provided by the International Entomopathogenic Bacillus Centre, Pasteur Institute, Paris (France).

The growth of B. thuringiensis thuringiensis 407 (pH 408) and B. thuringiensis kurstaki HD-73 strains, which synthesize Cry1Ab and Cry1Ac proteins, respectively, was carried out in Glucose medium, at 28±2ºC and 180 rpm, until 90% of cell lyses was achieved. The culture was centrifuged at 5000 rpm, 5ºC, for 15 min, and the gotten isolate was washed with phosphate buffer (0.1M NaH₂PO₄.H₂O + 0.1M NaCl, pH 6.0). Spores, crystals and cell traces separation was carried out through the application of bacterial suspension in sacarose gradient (67 - 79%), which was centrifuged at 9500 rpm, 5ºC, for one hour. Bands deposited among the various sucrose concentrations were collected, washed with mili-Q water and observed under phase-contrast microscopy. Next, proteins were solubilized in pH 10 phosphate buffer (50 mM Na₂CO₃, 10 mM DTT, 5 mM EDTA, 0.1 mM PMSF), following the method described by Fiuza et al. (14). Protein concentration was determined through the method described by Bradford (10), and the proteic profile was assessed in SDS-PAGE, at 10% (21).

The assays were performed at the Unisinos Microbiology Laboratory, where each fungal strain (Rhizoctonia solani, Pyricularia grisea, Fusarium oxysporum and Fusarium solani) was moved onto Petri plates, with platinnum loop, in PDA medium culture at a central site. Then, B. thuringiensis thuringiensis 407 (pH 408) and B. thuringiensis kurstaki HD-73 bacterial strains were inoculated by a platinnum loop striation, 1.5 cm far from where the fungus was previously inoculated, except for the control plates, with no bacteria. Micelial growth, at 28ºC, was assessed 7 and 14 days after incubation, by determining the fungal colony diameter. The experiments comprised 3 treatments and 3 repetitions for each fungal strain. Data went through Analysis of Variance and Tukey's test (P<0.05) for means comparison.

The assays were carried out at the Microbiology Laboratory, in Unisinos, where the antifungical activity of Cry1Ab and Cry1Ac proteins of B. thuringiensis for the previously mentioned 4 phytopathogens was determined through paper disk diffusion. The 10⁵spores /mL inoculum, was prepared for each phytopathogenic organism on plates with BDA medium. In the central portion of the plate, it was placed a filter paper disk (Æ = 10 mm) on the inoculum. Next, it was soaked at the maximum allowed concentration for each protein, corresponding to 2.4 µg for cry1Ab and 12 µg for Cry1Ac. These experiments comprised 3 treatments and 3 repetitions for each fungical strain, when it was measured the inhibition halo of the fungical growth. In order to ascertain the effect upon the conidiogenesis three random sites were cut on each plate, and each colony was immediately placed into a glass tube, with 9 mL of distilled water plus Tween 80 (0.1%), to be then agitated for about two minutes in vortex until the conidia were taken out of the surface of the cut medium. Conidia quantification was performed with the help of a Neubauer's chamber. Data went through Analysis of Variance and Tukey'S test (P<0.05) for means comparison.

B. thuringiensis strains which synthesize Cry1Ab e Cry1Ac proteins have decreased the micelial growth of Rhizoctonia solani,Pyrizularia grisea,Fusarium oxysporum and F. solani fungi during the assessment period when compared to the controls (Fig. 1).

Both strains have inhibited the phytopathogens growth tested up to seven days after incubation (Table 1), when there was a significant difference (P<0.05) between bacterial treatments and control groups.

The inhibiting effect of B. thuringiensis strains on phytopathogenic fungi breed can be associated with enzime production that can act against the fungal cell wall, since some bacteria antagonistic of phytopathogenic fungi bring about chitinases (3,26). In such context, Barboza-Corona et al. (6) have selected and characterized B. thuringiensis enzymes (chitinases) from Mexico, and have arrived at the conclusion that the synergistic action between chitinases and Cry proteins can be applied on phytopathogenic biological control.

Bettiol and Kimati (8) have also reported the occurrence of a large number of organisms antagonistic to Pyricularia oryzae Cavana, of which the most efficient ones belong to Bacillus genus. This endorses this study data, of which the tested strains were efficient against many phytopathogens. Endophytic bacteria, as the Pseudomonas, are also used as control agents against phytopathogenic fungi, such as F. oxysporum vasinfectum (11) and R. solani in the cotton culture (30), thus showing bacteria as potential biological control agents.

Bacillus sp. is used as a control agent, capable of bringing forth side antibiotic and metabolites (3,20), and an array of enzymes that degrade cell walls, such as amylases, glucanases, among other ones (15). Hence, Cho et al. (12) have tested Bacillus sp CY22 endophytic bacteria, isolated from the Platycodon grandiflorum root, and they have checked that Bacillus sp. CY22 makes the iturin A antibiotic, which has antifungical activity against R. solani,Phytium ultimum and F. oxysporum. That antibiotic can be associated with the inhibiting effect observed in this paper, because the used strains also belong to the Bacillus genus.

R. solani has high sensibility to B. subtilis, even in the smallest pathogen dose it was sensible (24,32). To acknowledge that bacteria efficacy, Bettiol e Lazaretti (9) have tested B. subtilis metabolites in bean seeds, effective to decrease the incidence of R. solani; similar effect of B. subtilis was not seen in rice seeds. As to the Fusarium spp fungus, the authors noticed its incidence decreased in rice seeds, although it has not showed positive results in bean seeds. Antagonistic bacteria, in general, with B. subtilis, act through symbiosis in a significant way, and, occasionally, through parasitism and competition (2). These results confirm this paper data, that is, the efficacy of Bacillus genus and the possibility of metabolites produced by the Bacillus genus to bring about the fungicidal effect.

A paper by Oshida et al. (28) showed that Bacillus amyloliquefaciens RC-2 filtrate culture has inhibited the growth of the Colletotrichum dematium (Persoon: Fries) fungus, besides other phytopathogens and bacteria, such as Rosellinia mecatrix,P. oryzae,Agrobacterium tumefaciens and Xanthomonas campestris pv. campestris. Mari et al. (25) suggest that the antifungical activity of bacteria, such as B. amyloliquefaciens, is due to a strive for nutrients. Those authors' observations ascertain similar interpretations for the effects of B. thuringiensis on the phytopathogens here assessed.

Works on the capacity of B. thuringiensis to deter the growth of phytopathogen fungi are scarce, but research by Batista-Junior et al. (7) stands out. They have tested a B. thuringiensis kurstaki HD1 strain which synthesizes Cry1 and Cry2 proteins, where Cry1Ab emerges as an inhibitor for the growth of F. solani,F. oxysporum and Colletotrichum sp. phytopathogens, thus confirming data from B. thuringiensis strains here assessed.

When the action of Cry1Ab and Cry1Ac proteins upon R. solani,P. grisea,F. oxysporum and F. solani was assessed, it was not noticed the development of the growth inhibition halo. Results obtained about conidia germination showed no significant difference (P<0.05) between the control and the treatments with Cry1Ab and Cry1Ac proteins on the previously mentioned fungi. These results can be related to the low proteic concentrations used in this study.

Data about Cry proteins are different from the ones about B. thuringiensis strains, which show positive effects on phytopathogenic fungi control. Because phytopathogens are not sensible to Cry proteins they can be associated with the production of other B. thuringiensis toxins, with low molecular weight (25-28 kDa), the so-called cytolitic toxins (Cyt), or the low proteic concentration assessed in this study, or other bacterial metabolites with no relationship with the proteic crystal. This is so because, besides crystal proteins, that bacterium is able to bring forth other toxins, such as the b-exotoxin, exoenzimes, and vegetative proteins or VIPs (17).

Batista-Junior et al. (7) have tested two strains: B. thuringiensis kurstaki HD1, which produces the crystal with insecticidal activity, and B. thuringiensis 407, which is a mutant one, which does not produce the proteic crystal against F. solani,F. oxysporum and Colletotrichum sp. phytopathogens. They managed to conclude that the absence of proteic cristal producer genes had not interfered with the degradation power of the micelium by the investigated B. thuringiensis isolates. That can confirm data of the present research, since the combination of spores, crystals and other toxins (VIP's, Cyt, b-exotoxina) brought forth by B. thuringiensis, was more effective, by inhibiting or controlling the growth of the investigated phytopathogens.

If we consider that under natural conditions B. thuringiensis spores remain on the soil for many years, various investigations have shown these spores do not germinate, and neither the cells multiply on the soil, thus decreasing the number of cells at 50% until sporulation (31,35). But the multiplication and conjugation of B. thuringiensis reach high indexes in dead insects' larvae (31). Also, the persistence of B. thuringiensis in the water have allowed us to notice that, in laboratory experiments, the cells and the spores can remain up to 10 days in this medium, with sporulation beginning between 12 and 15 hours after inoculation (16). In this context we advise the carrying out of field assays with the B. thuringiensis isolates used in this research, which can be useful in a biofungicidal formula similar to the work carried out by Vidhyasekaran et al. (34), who used Pseudomonas fluorescens to control Bacillus oryzae, with an activity equivalent to the chemical fungicide, which allowed for an increase in rice production.

It can be assumed that B. thuringiensis thuringiensis 407 (pH 408) and B. thuringiensis kurstaki HD-73 strains have inhibited the micelial growth of the phytopathogenic fungi R. solani,P. grisea,F. oxysporum and F. solani, while proteins at the tested concentrations had no effect. Hence, data from this study are promising regarding the usage of strains for the biological control of the tested fungi.

Submitted: November 24, 2006; Returned to authors for corrections: March 12, 2007; Approved: June 21, 2007

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Publication Dates

• Publication in this collection
  17 Oct 2007
• Date of issue
  Sept 2007

History

• Reviewed
  12 Mar 2007
• Received
  24 Nov 2006
• Accepted
  21 June 2007