Analele tiinifice ale Universitii Al. I. Cuza Iai s. II a.

Biologie vegetal, 2012, 58, 2: xx-xx

http://www.bio.uaic.ro/publicatii/anale_vegetala/anale_veg_index.html ISSN: 1223-6578, E-ISSN: 2247-2711

CHARACTERIZATION OF THE IMPACT OF BACILLUS LICHENIFORMIS AND PSEUDOMONAS AERUGINOSA AGAINST ALTERNARIA ALTERNATA BY PHASE CONTRAST MICROSCOPY AND TRANSMISSION ELECTRON MICROSCOPY Monica Elena MITOI1*, Florena Elena HELEPCIUC1, Aurelia BREZEANU1, Clina Petrua CORNEA2
Abstract: Pseudomonas and Bacillus are the most studied groups of plant disease-suppressive bacteria. Plant protection is mediated by the antagonistic effects of these bacteria against phytopathogens. The antifungal activity of several Pseudomonas spp. with highly degrading capacities, used in soil bioremediation, respectively some strains derived from Bacillus licheniformis, was established by dual test cultures with different fungal pathogen species as Alternaria alternata, Pythium debaryanum, Fusarium oxysporum and Botrytis cinerea. In early stages, Pseudomonas strains have a higher inhibitory activity on mycelium development, although the inhibition zone is more stable in time for Bacillus licheniformis. For the microscopic analyses, liquid media inoculated with Alternaria alternata, and simple or mixed culture of bacilli respectively pseudomonads were used. Light microscopy observations showed that pseudomonads tend to adhere along hyphae, while bacilli form clusters around hyphae. Transmission electron micrographs revealed that in interaction with bacilli, fungal cells release numerous electron-dense granules or vesicles with fibrilar content, while in the presence of pseudomonads, the fungal cell walls were coated by a network of microfilaments and fibrils. Most fungal cells have corrugated, very thick and heavily melanized cell walls, characteristic to the spores and are spatially separated by bacilli. In interaction with pseudomonads, fungal cells were surrounded by bacteria and presented detachment of the plasmatic membranes, coalescence of cytoplasm and degradation of intracellular membrane system. The destruction and disorganization of cell content in hyphae and conidia was also observed in the later stage of coculture with bacilli. The results indicate that the two types of bacteria have different mechanisms of action against fungal pathogen. Keywords: Alternaria alternata, antifungal activity, Bacillus, Pseudomonas, ultrastructure

Introduction The reduction of diseases incidence and severity in plants following soil amendment with some Pseudomonas and Bacillus strains was often observed (Benhamou et al., 2000). The importance of these two bacterial groups among the PGPBs (Plant GrowthPromoting Bacteria) is determined by the characteristics exhibited by these two genera. These bacteria have a wide range of biochemical equipments that contribute to their capacity to suppress pathogens. They have different mechanisms of action, both direct, through which bacteria influence the pathogens growth or the plant resistance, and indirect, through which bacteria stimulate the plant development, decreasing in this way the risk of infection. Recent progress in the identification of antifungal metabolites led to the idea that plant protection against fungal pathogens is provided in part by production of bacterial antibiotics associated with a possible competition for nutrients and iron in the rhizosphere
1

Institute of Biology, Romanian Academy, Department of Plant and Animal Cytobiology, Splaiul Independentei 296, 060031 Bucharest, Romania. e-mail: monica.carasan@ibiol.ro (corresponding author) 2 Faculty of Biotechnology, University of Agronomical Sciences and Veterinary Medicine, Bd. Mrti No. 59, 71331 Bucharest, Romania

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(Benhamou et al., 2000). Pseudomonads and bacilli have the capacity to colonize the rhizosphere and phylosphere (O`Sullivan and O`Gara, 1992), and to produce metabolites with antagonistic activity, such as antibiotics: 2,4 diacetylphloroglucinol, phenazyne, pyrrolnitrin, pyoluteorin, cyclic lipopeptides synthesized by Pseudomonas spp. (Brencic and Winans, 2005; Lee et al., 2003; Shanahan et al., 1992) or oomycin, zwittermicin A, kanosamine and lipopeptides produced by Bacillus spp. (Bais et al., 2004 ; Compant et al., 2005 ; Handelsman and Stabb, 1996). Moreover, these bacteria are able to excrete siderophores, such as pyoverdin and pyochelin produced by Pseudomonas spp. (Whipps, 2001) or to exhibit hyperparasitic activity, attacking pathogens by excretion of cell wall hydrolases like chitinases, -glucanases, laminarinases or proteases (Compant et al., 2005; Whipps, 2001). Most of the studies concerning the antifungal activity of some rhizobacteria are focused on the identification and purification of antibiotics, but little attention has been paid to the fungal responses to these compounds. Although the activity of antifungal compounds with chemically different structures is well documented, scarce data exist on the effects of these natural fungicides, on the morphology and ultrastructure of fungal pathogens (Romero et al., 2007; Souza et al., 2003; Thimon et al., 1995), and therefore, their mode of action remains unclear. Analyses of the antagonistic interactions at cellular level are not so numerous and focus on the relationships established between the plant, pathogen and antagonist (Benhamou et al., 2000; Cherif et al., 2003; El-Ghaouth et al., 1998). Recently, new microscopic techniques were developed for monitoring the relationships between the antagonist bacteria and plant pathogen in rhizosphere (Tombolini et al., 1999; Troxler et al., 1997) or to determine subcellular changes in the eukaryotic pathogen during interaction with the antagonistic prokaryote (Deora et al., 2006). Most studies on the sensitivity of plant-pathogenic fungi take into account only one species or isolate of antagonist bacteria and consider only one particular stage in the life cycle of a pathogen, usually mycelial growth (Souza et al., 2003). In this study, the inhibitory activity of selected strains of Pseudomonas aeruginosa with highly degrading capacities, used in soil bioremediation, and some strains obtained from Bacillus licheniformis by mutagenesis and transformation (Mateescu et al., 2003; Mateescu et al., 2005) was tested on fungal necrotrophic pathogens as Alternaria alternata, Pythium debaryanum, Fusarium oxysporum and Botrytis cinerea strains. The antagonistic reaction between the two partners was monitored by macroscopic and microscopic observations in different stages of cocultivation of selected bacteria with two Alternaria alternata strains. The direct contact of bacteria with mycelium and spores was observed by light microscopy and the cellular modifications induced by the antagonistic interaction between the two partners were studied on transmission electron micrographs. Materials and methods Biological material The bacterial strains used in this study were Pseudomonas aeruginosa (strains P7, P14, P18), Bacillus licheniformis (B40, tB40, mB40). Pseudomonads used in experiments are Romanian isolates from oil polluted areas that were identified and characterized using Biolog system and molecular techniques (ITS-RFLP) (Cornea et al., 2006). The Bacillus

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licheniformis strains were provided by the Faculty of Biotechnology, USAVM (Bucharest, Romania) being obtained in previous experiments (Mateescu et al., 2005). The inhibitory activity was tested on some nectrotrophic fungal pathogens such as Alternaria alternata (A1 and A100), Botrytis cinerea, Fusarium oxysporum and Pythium debaryanum. The pathogen strains were obtained from the Institute of Plant Protection (Bucharest, Romania). Culture media and conditions The bacterial strains were grown on LB medium, while fungi on PDA medium (Potato Dextrose Agar). PDA and Sabouraud medium (MS) were used for the interaction studies. The bacterial cells were cultured in simple and mixed variants, at 28C. The mixed variants were represented by cocultures of pseudomonads (P) or bacilli (B). Solid medium interactions The fungi and bacteria interaction studies were performed by juxtaposition at 3 cm distance from each other, on Petri dishes containing PDA solid medium with 1.5% (w/v) agar. The antagonist bacteria were placed at three equidistant sites (10 l of bacterial suspension in each spot) and the fungal pathogen (agar discs with 6mm diameter collected from Petri dishes with growing hyphae on PDA medium) was inoculated in the center of these sites. In the control cultures, bacterial suspensions were replaced with sterile LB medium. The inhibitory activity was calculated using the formula: % inhibition = (C S)/C X 100, where C represents the average of four replicates of mycelium extension (cm) in the control and S is the mycelium extension (cm) towards the bacterial colony (Lee et al., 2008). The measurements were made daily during 14 days of incubation at 28C in three replicates. Liquid medium interactions The fresh bacterial cultures were obtained by propagation on LB medium for 12 h at 28C. Liquid PDA medium was inoculated with 2% (v/v) of fresh bacterial suspension and actively growing mycelium of Alternaria alternata (strains A1 and A100, respectively). These cocultures were incubated at 28C, and the observations were made daily during 6 days. The samples were used for squash analyses under the light microscope and ultrathin sections were prepared for electron microscopy. Squash cytology Squash preparations were obtained by using several droplets of the mixed cultures, harvested between 1 and 6 days from inoculation. The samples represented by simple and dual liquid cultures were analyzed by phase contrast microscopy or contrasted with 4 (w/v) methylene-blue solution prior examination by light microscopy. Electron microscopy For electron microscopy, the samples were collected at 24 h, 3 and 5 days of cocultivation, respectively. The samples from cocultures and the simple or mixed cultures of the control were processed following the standard method (Mascorro and Bozzola, 2007), with some adaptations: samples were subjected to a prefixation in a solution of 3% (v/v) glutaraldehide in 0,1 M sodium cacodylate buffer, pH 7.2 at 4C, overnight, and rinsed with the same buffer. The biological material containing bacterial cells and fungal mycelium was incorporated in 2.5% (w/v) agar and then sectioned in 2 mm pieces. The agar included cells were washed several times with 0.05 M sodium cacodylate buffer, for 23 hours and then post-fixed in 1% (w/v) osmium tetraoxide solution in the same buffer, at 4C, overnight. After washing for 2 h with distilled water, the samples were dehydrated in a

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graded series of 10-100% (v/v) ethanol. The samples were washed twice with propylene oxide and finally embedded in Epon 812 resin. The samples were ultrasectioned at ultramicrotom (LKB, Sweden) with diamante knife and ultrathin sections were stained according to Reynolds double coloration (Reynolds, 1963), before examination with an EM-125 (Selemi, Ukraine) transmission electron microscope at 50 kV. Results Determination of the antifungal activity of bacterial strains The interactions on solid medium revealed the antagonistic effect of the bacterial strains used throughout this study on some fungal pathogens. The fungal mycelium had a better development on PDA medium than on the MS medium. However in dual cultures with bacilli, a more evident inhibitory action (clear zone of mycelial inhibition) was observed on MS medium. The dual culture tests demonstrated that the highest inhibitory activity was register in the fifth or sixth day of cocultivation with Alternaria alternata strains. The highest activity was detected for Bacillus licheniformis strains B40 and mB40 respectively, while the transformed bacteria (tB40) presented the lower activity against A1 and insignificant activity against A100. For the tested Pseudomonas strains, a high inhibitory activity was observed for P14, P18 and P7, but lower than B40 and mB40 strains (Table 1). In the case of Bacillus licheniformis, the inhibition zone was clearer than the one that appears in the presence of Pseudomonas, this aspect being correlated with a more stable and a higher inhibitory activity (Table 1). However, in early stages of the mycelium development, Pseudomonas strains restricted the circumferential growth of fungal colony, while, during co-cultivation with Bacillus licheniformis the inhibition of mycelium extension occurred only in the bacterial colony direction. It seems that Bacillus licheniformis strains excreted metabolites that act as a barrier between the fungi and bacteria, the mycelium development being restricted due to the synthesis of compounds with fungistatic activity surrounding colonies, while the Pseudomonas aeruginosa strains reduced the entire growth of mycelium by other mechanisms such as competition for nutrients. At the same time, the bacilli and pseudomonads can inhibit the fungal growth, by the excretion of lytic enzymes or other compounds with fungicidal activity. A different reaction was detected in the case of some fungal necrotrophic pathogens such as Pythium debaryanum, Botrytis cinerea and Fusarium oxysporum (Table 1), in the presence of the two species of bacteria. In the case of Pseudomonas strains, a high activity was observed in the first stages, but the activity decreased in time. Strains P14, P18 and the mixed pseudomonads had the highest activity on Pythium debaryanum, with a maximum in the fifth day, similarly with B40 and mB40 (Table 1). In the early stages of cocultivation with Pythium debaryanum and Botrytis cinerea the fungal mycelium was more developed in the presence of Bacillus licheniformis than Pseudomonas aeruginosa strains and only B40 and mB40 strains restricted the mycelial extension in later stages of dual cultures. In the particular case of Fusarium oxysporum the mycelium development was delayed and therefore, the inhibitory activity of bacteria increased in time, although the P7

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strain presented a peak of activity from the second day (Table 1). The color of the mycelium changed from pink to brown in the presence of Pseudomonas strains. An increased inhibitory activity was observed by mixing the bacterial strains belonging to the same genus, proving a synergistic action. When the inhibitory activity of the bacterial strains was tested on liquid medium, a poorly developed fungal mycelium, compared with the control, could be observed. Some differences between the fungal mycelium treated with bacilli or pseudomonads were detected. In the presence of Bacillus licheniformis strains, the fungal mycelium was more developed on the walls of the test tube, while in presence of Pseudomonas aeruginosa, the mycelium had a limited growth, at the surface of the medium. Moreover, in the case of Pseudomonas Alternaria cocultivation, the color of the medium became yellow and fluorescent under UV illumination, proving the synthesis of fluorescent pigments by the bacterial strains. Initially, the color appeared as a ring at the interface between the growth area of fungus and bacteria, and the coloration spread afterwards in the entire volume of the culture. The aspect of fungi and bacteria cocultures in light microscopy In the early stages of Pseudomonas aeruginosa and Alternaria alternata cocultivation, the fungal hyphae were rare and isolated from the bacterial colonies (Plate I e). These results could be due to the synthesis of some diffusible compounds which inhibited the fungal development. In coculture with bacilli, clusters among hyphae (Plate I f) and conidia formation (Plate I j) were observed. After 5 days from inoculation, in the case of the samples with Pseudomonas aeruginosa, the bacterial cells tended to adhere to the fungal hyphae (Plate I g and h), while Bacillus licheniformis cells were agglomerated along the hyphae (Plate I k and i). The bacterial pellicles included the fungal hyphae (Plate I i). In the later stages of cocultivation with Alternaria alternata, both Bacillus licheniformis and Pseudomonas aeruginosa strains seem to have direct contact with the fungal hyphae, probably due to different antagonist mechanisms. The ultrastructure of Alternaria alternata in interaction with antagonist strains of bacilli and pseudomonads Observations of the fungal cell ultrastructures, in the control sample, shown different stages of life cycle from young hyphae and spores to mature hyphae, but mature forms of conidia were not observed (Plate II a-e). The sections revealed a high density of spores and hyphae, frequently agglomerated in variable disposals (Plate II a and b). The ultrastructure of fungal cells displays the densely cytoplasmic contents with large quantity of storage products such as lipids and vacuoles with distorted (irregular) forms (Plate II a and d). The walls have a fibrous structure with two ill-defined layers with a granular electron-dense substance concentrated in the outer region. The thickness of cell walls and deposition of dark pigment, probably melanin on primary cell wall varied with the age. The micrographs of control variants of the antifungal bacterial strains showed that Pseudomonas strains released electron-dense material and vesicles of different size in the extracellular medium (Plate II f). This material might be involved in the production of the extracellular polymeric matrix of bacterial biofilm-like structures, such as pellicles in the liquid media. In dual cultures, the bacterial vesicular production was more intense, both in bacilli and pseudomonads cocultures, although the presence of fungal hyphae was not observed in

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the sections (Plate II h). Most of bacteria, including Pseudomonas aeruginosa and more species of bacilli, are able to form biofilm. This multicellular mode of growth predominates in nature as a protective mechanism against hostile conditions (Walker et al., 2004). Ultrastructural modifications of fungal cells at 3 days of cocultivation In the case of cocultures with bacilli, most of the sections showed only fungal or bacterial cells, this observation leading to the conclusion that bacterial population are spatially separated from the fungal mycelium, a characteristic that has been observed in the liquid medium as well. The fungal primary cell walls had an irregular shape (Plate III e) with verruculose protuberances on the outer surface (Plate III c). Different sized vesicles with fibrilar content are detached from these cell walls (Plate III a and b), as a possible response to the bacterial action, in particular to the bacterial synthesis of lytic enzymes. The agglomeration of bacterial cells around the fungal hyphae was observed when the section crossed an area with both fungal and bacterial cells. The response of fungal cells to the action of bacteria was different, depending on the bacterial species. When P14 strain was used, most of the fungal cells appeared surrounded by a fibrilar layer exhibiting different degrees of thickness (Plate IV a and b). This barrier layer between hyphae and surrounding bacteria was disintegrated (Plate IV c) and most fungal cells presented a detachment of the plasmalemma from the cell wall (Plate IV d and e). Other structural modifications observed in the bacterial-fungal interaction were: the contraction of cytoplasm (Plate IV f and g), secretion of extraprotoplasmic vesicles and degeneration of membrane system (Plate IV g), phenomena characteristic for cell plasmolysis. In severe cases, plasmalemma was disrupted and cytoplasm appeared to lose its structural organization (Plate IV f). The presence of bacteria induces a premature senescence and further autolysis and disorganization of hyphae cytoplasm, resulting in ghost cells empty of content. Ultrastructural modifications of fungal cells at 5 days of cocultivation After 5 days of coculture, in the presence of the bacterial strain B40 mature conidia with thick ornamented cell walls were detected (Plate III f and g). These thickened cell walls were electron-dense, probably due to the melanin deposition. Moreover, in this sample, fungal cells seemed to release large melanized particles, comparable with bacterial cells dimensions (Plate III f). In this stage of fungal cocultivation with mixed culture of Bacillus licheniformis some hyphae and mature conidia showed a degraded cell content with membrane shrinkage, irregular contour of cytoplasm, that included multiple vesicles (Plate III i and j). Additionally, at 5 days, other changes were detected: spores and few vegetative hyphae with more thickened cell wall (Fig 5 j and h), rare conidiophores in germination (Plate IV i) and formation of endospores (Plate III e). These modifications suggest that in latter stages of cocultivation the vegetative growth was inhibited and conidia development ceased both in the presence of bacilli and pseudomonads. Discussions The selected bacterial strains, tested in our experiments, showed inhibitory activity on a broad range of necrotrophic pathogens, including Alternaria alternata, Botrytis cinerea, Pythium debaryanum and Fusarium oxysporum. Apparently, the two types of bacteria have different mechanisms of action. Initially, Pseudomonas aeruginosa strains

Mitoi, M.E. et al., 2012/ An. Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 58, 2: xx-xx

had a higher inhibitory activity, the growth of fungal mycelium being reduced, while the antagonistic activity of Bacillus licheniformis was more stable in time. Pseudomonas aeruginosa strains produced more diffusible compounds than Bacillus licheniformis, restricting the mycelium development from the beginning. At the same time, it is possible that B40 strain excreted active molecules with longer lifetime or with a different mode of action, maintaining the inhibitory zone observed around the bacterial colony. Recently, an antagonist lipopeptide was identified from culture filtrate of Bacillus licheniformis BC98 (Tendulkar et al., 2007). The release of fluorescent pigments by Pseudomonas aeruginosa strains varies with antifungal activity, thus the culture media of P14 and P18 strains emitted a high fluorescence, while P7 had a lower fluorescence signal. Based on the literature (O`Sullivan and O`Gara, 1992), the yellow fluorescent color appears in the culture medium as a result of pyoverdine production, which is a specific siderophore for fluorescent pseudomonads. The iron starvation conditions determined by the excretion of these iron-binding ligands, probably, prevent the germination of fungal spores. The transmission electron microscopy micrographs showed that in the presence of P14 strain, fungal cells were surrounded by many filaments and vesicles. The vesicles may be secreted as a response of antagonistic reaction between the two partners. The filamentous network on the outer surface of the cell wall could have a protective role against bacteria or could be a result of destabilization in the cell wall structure. Thus, the fungal cells which present a detachment of the plasmatic membrane have a diminished filamentous network and bacterial cells are found much closer to the fungal cell wall. In the control samples, this coat of fungal cell wall was not observed, thought in classical utrastructural description of mature hyphae the walls are covered with a loose membranous layer (Campbell, 1970). A major change in the structure of fungal cells consisted of the degeneration of membrane system. These results provide the idea that the active substances produced by P14 strain affected the membrane system of the fungus similar with other fungi exposed to synthetic fungicides (Youssef and Koblett, 1998). A common mode of action of these diverse fungicides is the inhibition of ergosterol biosynthesis, a key sterol compound of fungal plasma membranes. Localized alterations in plasma membrane organization, a degenerated cytoplasm bordered by retracted plasma membrane were observed also in hyphal tips exposed to 2,4- diacetylphloroglucinol (Souza et al., 2003), one of the most studied compounds with antifungal activity produced by Pseudomonas spp. In the presence of B40 strain, most of the fungal cells had very thick, heavily melanized, ornamented cell walls, characteristic to mature conidia, suggesting that only in this particular stage of the life cycle, the plant pathogen is resistant to bacteria. Cell walls of numerous fungi are melanized, melanin protecting the pigmented cell from physical and biological stress by forming a physical barrier between the cell and its surroundings. Conidia of Alternaria alternata presented thicker cell walls produced by deposition of fibrilar material encased in an electron-opaque layer. It was shown before that melanin deposition occurs from the time of spore formation, in very last stages of maturation and primary walls become progressively more electron-dense and completely opaque on the outer surface (Campbell, 1969). From many years, significance of melanin in survival of spores was appreciated, because these structures can be degraded without melanin protection (Butler et al., 2001). In our case, the detachment of electron-dense particles or

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vesicles with fibrilar content from spore or hyphal cell wall could be a defensive mechanism, but also the result of wall-degrading enzymes produced by B40 strain. The melanins can protect fungal cell walls components against degradation by inhibition of lytic enzymes as chitinases and glucanases. Also, the melanin may provide protection against fungicide by its permeability barrier function or by sequestration of antifungal peptides (Butler et al., 2001). Most of the analyzed ultrathin sections showed that, in coculture with bacilli, the bacterial and fungal cells were, in major part, spatially separated. However, the fungal cell wall modifications were observed both in the areas where bacteria interact with fungi and in the sections that present only fungal cells. The main conclusion that can be derived from this observation is that bacteria do not necessarily need to be in direct contact with fungal cells in order to induce modifications at cellular level. Conclusions Considering the observations mentioned above, we concluded that fungal cells react different in the presence of the two types of bacterial strains. The main structural modifications at the cell wall level were the apparition of protuberances, release of vesicles or electron-dense particles, in the case of bacilli, while cocultivation with pseudomonads determined the formation of fibrous layers on the outer surface of the fungi cells. Drastic modifications, which can indicate a lytic effect of P14 antagonistic bacteria against fungi, like plasmalemmal detachment, constriction of cytoplasm and degradation of membrane system appeared quite often as well. Typical ultrastructural changes observed in fungi exposed to fungicides were also detected in coculture with Bacillus licheniformis strains, both in hyphal tips and mature conidia, but not very often. Probably these bacteria produce a mixture of compounds with multiple mechanisms of action from inhibition of vegetative growth to massive destruction and disorganization of fungal cells. Undergoing studies approach the identification of genomic sequences in our bacilli and pseudomonads strains responsible for the synthesis of antifungal compounds which are commonly produced by bacteria from Bacillus and Pseudomonas genus. Acknowledgments This study was supported with funds from National Center of Programs Management (CNMP), Ministry of Education and Research, Romania (CEEX 52/2006- BIOCOMB project). We thank Brnzan A. and Stan V. for technical assistance. REFERENCES
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Youssef, N.N., Knoblett, J., 1998. Culture filtrate of Bacillus atrophaeus induced abnormalities in Ascosphaera apis. Mycologia. 90, 6: 937-946.

PLATE I. Squash analyses of Alternaria alternata and selected strains of Bacillus licheniformis or Pseudomonas spp. cultures: (a) and (d) morphological aspects of hyphae in control variants; the aspect of bacilli (b) and pseudomonads (c) in phase contrast microscopy; early stages in interactions between fungi and bacteriapseudomonads forming biofilms (e) and bacilli clustering among hyphae (f); in the latter stages of interaction the pseudomonads adhere along hyphae (g and h) and bacteria biofilm invaded fungal hyphae (i), while bacilli clustering around hyphae (k and l), but conidia germination was not inhibited (j). Scale bars = 100 m.

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PLATE II. Ultrastructural aspects of Alternaria alternata, Bacillus licheniformis and Pseudomonas aeruginosa in control samples. Sections through hyphae and spores with different sizes and shapes ( a-b), longitudinal section (d) and cross section (c) by vegetative hyphae with cytoplasmic organelle densely packed, large inclusion of the storage products (SB), irregular shape of vacuole (Va) and cell walls (CW) relatively smooth and spore with thick cell walls and rough surface (e). Scale bars = 1 m. Bacterial liquid cultures of Pseudomonas aeruginosa (f) and Bacillus licheniformis (g) show production of vesicular material (f- see arrows) and release of large quantity of electron-dense material in dual cultures (h). Scale bars = 0.5 m.

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Mitoi, M.E. et al., 2012/ An. Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 58, 2: xx-xx

PLATE III. Transmission electron micrographs of Alternaria alternata fungal cells in interaction with Bacillus licheniformis strains. (a-e): release of vesicles with fibrilar content from the conidia (a and b) and hyphae (c) cell walls, detachment of the melanized granules from conidia cell walls ( d and f- see arrows) and irregular thickness of cell wall (e), mature conidia with corrugated, very thick and melanized cell walls ( f and g), the presence of endospore (h) and mature conidia and hyphae with constricted cytoplasm which contains multiple vesicles (i and j). Scale bars = 1m.

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Mitoi, M.E. et al., 2012/ An. Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 58, 2: xx-xx

PLATE IV. Transmission electron micrographs of Alternaria alternata fungal cells in interaction with Pseudomonas aerugiosa strains. Hyphae surrounded by bacteria, with a fibrilar network around the cell wall ( a and b), detached plasmalemma from the cell wall (d - see arrow and e) loss of fibrilar network (c-arrows and e), shrinkage of cell content (c, f and g), degradation of membrane system (f and g), and secretion of extraprotoplasmic vesicles (g- see arrow). Hyphae and conidia with thicker cell walls (h and j), cell almost depleted of content (h and j) and rare conidiophores with abnormal germination (i). Scale bars = 1m.

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Table 1. Inhibitory activity of Bacillus licheniformis and Pseudomonas aeruginosa strains against different fungal phytopathogens
Phytopathogens Bacterial strains P7 P14 P18 B40 mB40 tB40 P7 P14 P18 B40 mB40 tB40 P7 P14 P18 B40 mB40 tB40 P7 P14 P18 B40 mB40 tB40 P7 P14 P18 B40 mB40 tB40 3 days 26.66 4.7 32.77 5.8 33.33 4.7 27.22 0.9 28.33 1.6 ns ns ns ns ns ns 25.68 6.8 32.24 1.8 (a) 32.24 2.5 (a) 28.96 3.7 (a) ns (a) ns ns ns 31.29 2.3 ns ns ns ns 4 days 31.54 1.8 41.83 4.1 41.61 0.9 39.14 3.0 40.04 0.7 ns ns ns ns ns 20.34 1.4 20.34 1.4 (a) 46.32 1.9 (a) 41.99 3.9 (b) ns (b) 32.33 3.4 39.30 1.7 36.31 1.7 50.24 2.2 44.77 6.3 ns ns ns ns ns 26.85 8.2 5 days 33.92 3.3 44.54 3.1 (a) 43.36 3.3 (b) 40.60 0.6 (b) 41.00 1.1 (b) ns (b) ns ns ns ns ns ns ns ns 46.58 1.4 41.45 4.5 (b) ns (b) 36.30 44.79 2.2(a) 40.55 5.3(a) 57.53 1.9 1.4(a) 52.22 4.5 (b) 34.18 4.0 (b) ns (a) ns ns ns 36.12 10.2 Inhibitory activity (%) 6 days 7 days 8 days 18.58 1.6 42.57 2.7 37.85 8.2 29.99 8.2 38.64 3.3 ns ns 38.64 2.0 37.85 1.3 37.06 1.8 39.42 1.3 39.82 1.1 39.03 1.8 ns ns ns 29.74 13.8 25.33 26.05 ns ns ns 11.5 11.8 ns 14.00 4.0 17.32 4.3 ns ns ns 7.52 2.1 ns 18.48 1.0 ns ns ns ns 46.58 1.4 45.72 1.9 46.15 2.2 41.45 4.5 40.17 4.1 41.45 4.5 32.28 8.7 ns ns ns 43.15 2.8 41.48 3.8 38.97 0.7 36.05 3.7 34.37 5.0 57.78 1.4 49.84 54.44 0.7 51.09 5.3 47.96 4.4 48.58 5.3 16.8 29.78 6.6 24.76 2.5 25.60 3.8 21.77 6.4 ns 31.11 1.7 27.18 1.7 25.21 1.7 (b) 30.62 4.4 30.13 6.8 19.80 2.2 (b) 43.91 2.0 43.91 2.0 43.17 1.0 (b) 45.38 8.9 43.91 5.1 41.94 3.0 (b) ns (b) 9 days ns ns 37.46 1.7 37.85 1.3 28.45 8.6 20.32 16.53 4.0 10.1 ns 21.40 1.8 ns 45.29 2.6 41.45 4.5 ns 39.81 4.3 33.12 54.44 0.7 5.2 48.58 5.3 21.42 3.8 23.24 2.9 16.35 4.5 43.17 1.0 42.43 3.9 10 days ns ns 37.46 37.85 0.9 1.1 27.27 22.22 6.5 19.19 7.9 23.23 1.8 27.27 8.4 ns 1.2 45.29 41.45 2.1 3.6 ns 37.30 32.70 3.6 51.93 4.1 47.33 0.5 20.58 2.5 2.3 23.24 14.39 2.4 43.17 2.4 42.43 0.7 3.1 13 days ns ns 37.06 37.85 1.8 1.3 27.02 26.12 9.3 20.72 7.8 ns 6.8 36.03 ns 1.5 45.72 43.58 2.9 4.4 ns 35.63 28.94 3.8 50.26 3.8 47.96 0.7 ns 4.4 20.29 ns 2.9 43.17 41.45 1.0 3.7 14 days ns ns 37.06 1.8 37.85 1.3 26.25 12.0 25.83 10.4 (ab) 23.33 10.1 (ab) 29.58 10.1 41.25 (a) 0.0 (b) (ab) 27.5 10.8 (ab) 45.72 2.9 43.58 4.4 ns 35.63 3.8 28.94 3.8 50.26 0.7 47.96 4.4 ns 20.29 2.9 ns 43.17 1.0 41.45 3.7 -

Pythium debaryanum

Fusarium oxysporum

Botrytis cinerea

Alternaria alternata A1 strain Alternaria alternata A100 strain

2 days ns ns ns ns ns 40.74 ns 9.8 ns ns ns ns ns ns ns ns -

- no activity; ns - no significant; data- significantly different than control (p<0.05); highlighted values = high and relatively stable inhibitory activity; values within the same column followed by same letters are not significantly different (p>0.05).

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