FUNGAL AND BACTERIAL GLYCOCONJUGATS INTERACTIONS: SPECIFIC MECHANISM BETWEEN LECTIN PRODUCING FUNGI

AND BACILLUS BIOCONTROL STRAINS?

Oana-Alina SICUIA1, C lina-Petru a CORNEA2, Aneta POP3

1Research and Development Institute for Plant Protection, 8 Ion Ionescu de la Brad Blvd., 013813, Bucharest, Romania, tel. 004-021-2693231, 33, 34, 36, fax. 004-021-2693239, e-mail:

sicuia_oana@yahoo.com; 2.University of Agronomic Sciences and Veterinary Medicine of Bucharest, Faculty of

Biotechnologies, 59 M r ti Blvd, 011464, Bucharest, Romania, tel. 004-021-318 36 40, fax. 004-021-318 25 88, e-mail: pccornea@yahoo.com

3University of Agronomic Sciences and Veterinary Medicine of Bucharest, Faculty of Veterinary Medicine, 105 Splaiul Independentei, 050097, Bucharest, Romania

Corresponding author: pccornea@yahoo.com

Abstract The mechanisms involved in the antagonistic action of biocontrol agents are intensively studied and several types were identified. A special attention was accorded to Bacillus strains that exhibit broad host spectrum against fungal pathogens. By studying in vitro interaction between some Bacillus biocontrol strains and some plant pathogenic fungal strains belonging to Botrytis cinerea, Penicillium sp. and Sclerotinia sclerotiorum species, a precipitation line was observed at the inhibition area level. This outcome was detected in co-cultivation of the biocontrol bacteria with the mentioned fungal species described as specific lectins producers, but not when strains of Alternaria, Fusarium or Pythium were used. The present study included in vitro co-cultivation of the biocontrol strains with B. cinerea on PDA medium supplemented with different carbon sources, sugars that are known to be interacting with different kind of carbohydrate-binding proteins. Significant differences in bacterial growth limitation were observed when the precipitation line was more evident and a reduced inhibition of the fungal growth was registered. Based on the fact that carbohydrate-binding proteins, also referred as lectin or agglutinins, have functions in defense responses to pathogen invaders, two hypotheses may be issued: there could be a chain reaction by which the biocontrol bacteria induces a defense mechanism in some fungi, and the lectin or lectin-like producing fungi reacts in self-defense and bind the nutrients so that the antagonistic bacteria are limited in growth and that certain substrates, enriched with particular carbohydrates, facilitate the activation of some defense mechanism in fungi against some antagonistic bacteria“, as a possible consequence of the low bacterial glycoproteins specificity for those carbohydrates, compared with the lectins or lectin-like compounds of some fungi that can precipitate the carbohydrate nutrients as defense mechanism against antagonistic bacteria, similar to the nutritional competition.

Key words: antagonistic bacteria, lectin producing fungi. INTRODUCTION Some filamentous fungal species have been reported to have the ability to produce lectins (Candy et al., 2001; Cornea et al., 2009; Inbar and Chet, 1994; Kellens et al, 1992). Lectins are associated various interactions among microorganisms (Ni and Tizard, 1996). Lectins are sugar binding proteins or glycoproteins, which agglutinate cells and/or precipitate glycoconjugates (Barak and Chet, 1990; Lam and Ng, 2011).

Among the filamentous fungi which are capable to produce lectins are mentioned various soil-borne and plant pathogens such as: Botrytis cinerea (Kellens et al, 1992), some Penicillium species (Singh et al., 2009), Rhizoctonia solani (Candy et al., 2001; Hamshou et al., 2007) and different members of the Sclerotiniaceae (Inbar and Chet, 1994; Kellens et al., 1992; Sulzenbacher et al., 2010) and some entomopathogenic fungi like Beauveria bassiana (Kossowska et al., 1999),

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AgroLife Scientific Journal - Volume 2, Number 1, 2013

ISSN 2285-5718; ISSN CD-ROM 2285-5726; ISSN ONLINE 2286-0126; ISSN-L 2285-5718

Conidiobolus obscurus (Latgé et al., 1988), Paecilomyces japonica (Park et al., 2004). Regarding the antagonistic interactions between fungi, it was found that one of the mechanisms used by mycoparasitic Trichoderma species in the biological control of the phytopathogens is the lectin-mediated recognition of the fungal hosts (Inbar and Chet, 1992, 1994, 1995). Thus, the phytopathogenic fungi that are lectins producers trigger the expression of the hyperparasitic character of T. harzianum biocontrol fungi (Inbar and Chet, 1994; Matroudi et al., 2009; Verma, 2009). It was shown that using Lectin-Coated Nylon Fibers (LCNF) induced the mycoparasitic character in the biocontrol fungus T. harzianum that produced hooks and coiled around the LCNF, in a pattern similar to that observed with the real host hyphae of Sclerotium rolfsii (Inbar and Chet, 1992). These findings provide direct evidence for the role of microbial lectins in mycoparasitism. In the hyperparasitic process a cell to cell recognition is needed between the lectin producig fungi and the mycoparasitic cells of Trichorerma (Inbar and Chet, 1992). However, if we consider the relations between lectin producing fungi and Bacillus biocontrol strains, the interaction is not direct, cell to cell contact. The communication is indirect and it is made by the secreted extracellular compounds (like lipopeptides or enzymes) that diffuse into the environment (Frey-Klett et al., 2011). The aim of this study was the analysis of bacterial and fungal glycoconjugates interaction as a specific mechanism between lectin producing phytopathogenic fungi and some bacterial biocontrol strains. MATERIALS AND METHODS Bacillus biocontrol strains The bacterial strains used in this study are Bacillus pumilus OS15, B. amyloliquefaciens OS17 and B. amyloliquefaciens BW. These three strains were previously characterized for their beneficial attributes in plant protection [Constantinescu et al., 2010; Dinu et al., 2012; Sicuia et al., 2011, 2012a,b]. Routinely, these strains are grown on LB agar medium at 28°C and maintained at 4°C.

Fungal material The phytopathogenic fungi used in this study (Table 1) were maintained on potato-dextrose-agar.

Table 1. Filamentous fungal species Phytopathogenic

fungi Provenience

Alternaria sp. RDIPP - Bucharest collection Botrytis cinerea RDIPP - Bucharest collection

Botrytis cinerea Faculty of Biotechnology Bucharest collection

Fusarium oxysporum RDIPP - Bucharest collection Pythium debarianum DSM 62946 DSMZ Collection, Germany

Penicillium sp. Faculty of Biotechnology Bucharest collection

Sclerotinia sclerotiorum RDIPP - Bucharest collection

Antagonistic interaction The antagonistic interaction between the phytopathogenic fungi and Bacillus biocontol strains were revealed on PDA medium, using the dual culture technique. The test was performed in Petri dishes where the fungi were placed in the middle of the plate and the bacterial strains were inoculated at 2 cm distance from the fungi. The fungal inoculums consisted in mycelia disc of 5 mm diameter from 7-day-old cultures. The bacterial inoculums used for the test was taken from fresh cultures. The plates were incubated at 28°C for 5-7 days. The interactions between fungal and bacterial glycoprotein were emphasized by the appearance of a specific precipitation line between the tested microbial strains, in different culture conditions: on PDA and PDA supplemented with 2% galactose or 2% raffinose. The co-inoculation procedure was similar to that mentioned above. RESULTS AND DISCUSSIONS During the analysis of antagonistic interactions between the selected biocontrol Bacillus strains and some phytopathogenic fungi a precipitation line was observed in the area of fungal growth inhibition. The precipitation line appeared only in the interactions with Botrytis cinerea (Figure 1a) and Sclerotinia sclerotiorum (Figure 1b). Typically, the location of this line of precipitation was closer to the edge of the antagonistic bacterial colony.

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Figure 1. The precipitation lines near the antagonistic bacterial strains Bacillus pumilus OS15

generated by Botrytis cinerea (a) and Sclerotinia sclerotiorum (b)

The presence of the precipitation lines in the area of interaction between bacteria and filamentous fungi has also been reported by other authors (Cornea et al., 2009, Machado et al., 2010) but their significance still remains unclear. Among the fungal species tested in these experiments, Botrytis cinerea and Sclerotinia sclerotiorum were previously described as lectin producing microorganisms (Cornea et al., 2009; Li and Rollins, 2010). Moreover, strains of Rhizoctonia solani (Hamshou et al., 2007; Cornea et al., 2009), Sclerotium rolfsii (Barak and Chet, 1990; Verma, 2009), Aspergillus sp. (Singh et al., 2008), and Penicillium sp. (Francis et al., 2011) were also mentioned as lectins producers. Our experiments prove that the presence of precipitation line is dependent of both fungal strain and antagonistic bacteria. Thus, clear precipitation line was observed only in co-cultivation of some known lectin producing fungi (Botrytis cinerea, Sclerotinia sclerotiorum) with certain bacterial strains. No such aspects were detected when Alternaria sp., Fusarium oxysporum or Pythium debarianum were used in interaction, despite their growth inhibition induced by the same bacterial strains. These aspects suggest that the precipitation is due to the interaction between lectin or lectin-like compounds secreted by mentioned fungi and bacterial glycoproteins or other compounds (possible lipopeptides). This aspect is sustained by a precipitation line between one strain of Penicillium sp. and B. pumilus OS15 (Figure 2). These aspects lead to hypothesis that fungal lectins may be released in the environment as a response to antagonistic bacteria specific signals.

Figure 2. The wide precipitation line generated by

Penicillium sp. near the growth of antagonistic Bacillus pumilus OS15 bacterial strains

The presence of lectins may lead to the retardation of inhibitory action of bacteria by agglutination of bacterial extracellular antifungal agents. This supposition is supported by the previously reported data about some bacterial proteins, located mainly at cell surface, that are glycoproteins (Messner, 2004; Nothaft and Szymanski, 2010). In this view, the precipitation line observed could be due to specific interaction between fungal lectins (carbohydrate-binding proteins) and the carbohydrate residues from specific bacterial glycoproteins. The involvement of fungal carbohydrate-binding proteins in microbial interactions was examined by in vitro co-cultivation of the selected biocontrol Bacillus strains with two strains of B. cinerea on PDA medium supplemented with galactose and raffinose, respectively. After comparative tests the differences observed were not only in the formation of precipitation lines (as expected due to the affinity interaction) but also in the size of the bacterial colony (Figure 3 a, b). Significant differences in bacterial growth limitation were observed when the precipitation line was more evident and a reduced inhibition of the fungal growth was registered.

a b

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PDA PDA + galactose PDA + raffinose

a Botrytis cinerea strain from RDIPP collection

PDA PDA + galactose PDA + raffinose

b Botrytis cinerea strain from the Faculty of Biotechnology collection USAMV Bucharest Figure 3. Interaction between Botrytis cinerea and the antagonistic bacterial

strain Bacillus amyloliquefaciens OS17

In the interactions between B. amyloliquefaciens OS17 strain and B. cinerea (RDIPP collection) on PDA medium no precipitation line occurred, but when PDA was supplemented with galactose thin precipitation line appeared and the bacterial growth was reduced. When the PDA was supplemented with raffinose the precipitation line was more abundant. Moreover the bacterial growth was significantly inhibited and the colony was much smaller (Figure 3a and 3b). Another noticed aspect was that the fungal growth was less inhibited when the medium was enriched with raffinose comparative with PDA with galactose or standard PDA medium. These findings suggest a possible stimulating effect of raffinose for the protective compound secreted by the fungi, and/or the inhibition of specific soluble bacterial compounds. These statements reflect a self defense mechanism in the phytopatogenic fungi against the biocontrol bacteria, possible mediated by lectins that are specific for glycoconjugates of these antagonists. However, it must be mentioned that even if the biocontrol activity is diminished the clear inhibition zones are still maintained, suggesting that the self protection mechanism developed by fungi is not absolute. Similar aspects were also registered for the other two biocontrol strains, Bacillus pumilus

OS15 and B. amyloliquefaciens BW but at lower intensity compared with B. amyloliquefaciens OS17 strain. The obtained results suggest that could be a chain reaction by which the biocontrol bacteria induces a defense mechanism in some fungi, based on lectin or lectin-like compounds, and/or bacteria produce specific soluble glycoconjugates either glycoproteins or lipopolysaccharides involved in antagonistic properties and in colony development. CONCLUSIONS In vitro interaction between the three Bacillus biocontrol strains and the phytopathogenic fungi Botrytis cinerea, Penicillium sp. and Sclerotinia sclerotiorum revealed a precipitation line at the inhibition area level. This aspect was attributed to the specific binding of sugar residues from bacterial glycoconjugates by the lectin or lectin-like compounds secreted by the fungi analysed. The presence of raffinose in the culture medium induced an intense precipitation line near the bacterial colony edge, suggesting a stimulation bacterial extracellular compounds production. The increase of the intensity of precipitation line is accompanied by the inhibition of

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