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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 BACI LLUS LI CHENIFORMI SAND

PSEUDOMONAS AERUGI NOSA AGAINST ALTERNARIA ALTERNATA BY

PHASE CONTRAST MICROSCOPY AND TRANSMISSION ELECTRON

MICROSCOPY

Monica Elena MITOI1*, Florena Elena HELEPCIUC1, Aurelia BREZEANU1, ClinaPetrua 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. Theantifungal activity of several Pseudomonas spp. with highly degrading capacities, used in soil

bioremediation, respectively some strains derived from Bacillus licheniformis, was established by dualtest cultures with different fungal pathogen species as Alternaria alternata, Pythium debaryanum,

Fusarium oxysporum andBotrytis cinerea . In early stages,Pseudomonas strains have a higher inhibitory

activity on mycelium development, although the inhibition zone is more stable in time for Bacilluslicheniformis. For the microscopic analyses, liquid media inoculated withAlternaria alternata, and simpleor 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. Transmissionelectron micrographs revealed that in interaction with bacilli, fungal cells release numerous electron-densegranules or vesicles with fibrilar content, while in the presence of pseudomonads, the fungal cell wallswere coated by a network of microfilaments and fibrils. Most fungal cells have corrugated, very thick andheavily melanized cell walls, characteristic to the spores and are spatially separated by bacilli. Ininteraction 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

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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

(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 metaboliteswith antagonistic activity, such as antibiotics: 2,4 diacetylphloroglucinol, phenazyne,

pyrrolnitrin, pyoluteorin, cyclic lipopeptides synthesized by Pseudomonas spp. (Brencicand Winans, 2005; Lee et al., 2003; Shanahan et al., 1992) or oomycin, zwittermicin A,

kanosamine and lipopeptides produced byBacillus spp. (Bais et al., 2004 ; Compant et al.,

2005 ; Handelsman and Stabb, 1996). Moreover, these bacteria are able to excretesiderophores, such as pyoverdin and pyochelin produced by Pseudomonas spp. (Whipps,

2001) or to exhibit hyperparasitic activity, attacking pathogens by excretion of cell wallhydrolases like chitinases, -glucanases, laminarinases or proteases (Compant et al., 2005;Whipps, 2001).

Most of the studies concerning the antifungal activity of some rhizobacteria arefocused 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 compoundswith 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 ofaction 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) t d t i b ll l h i th k ti th d i i t ti

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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 Pythiumdebaryanum. The pathogen strains were obtained from the Institute of Plant Protection

(Bucharest, Romania).

Culture media and conditionsThe bacterial strains were grown on LB medium, while fungi on PDA medium

(Potato Dextrose Agar). PDA and Sabouraud medium (MS) were used for the interactionstudies. The bacterial cells were cultured in simple and mixed variants, at 28C. The mixedvariants were represented by cocultures of pseudomonads (P) or bacilli (B).

Solid medium interactionsThe 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 collectedfrom Petri dishes with growing hyphae on PDA medium) was inoculated in the center ofthese sites. In the control cultures, bacterial suspensions were replaced with sterile LB

medium. The inhibitory activity was calculated using the formula: % inhibition = (CS)/C

X 100, where C represents the average of four replicates of mycelium extension (cm) in thecontrol 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.

Li id di i t ti

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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 bacterialstrains 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) wasobserved on MS medium.

The dual culture tests demonstrated that the highest inhibitory activity was register inthe fifth or sixth day of cocultivation withAlternaria alternata strains. The highest activity

was detected forBacillus licheniformis strains B40 and mB40 respectively, while thetransformed bacteria (tB40) presented the lower activity against A1 and insignificant

activity against A100. For the tested Pseudomonas strains, a high inhibitory activity wasobserved for P14, P18 and P7, but lower than B40 and mB40 strains (Table 1). In the case

ofBacillus licheniformis, the inhibition zone was clearer than the one that appears in the

presence ofPseudomonas, this aspect being correlated with a more stable and a higher

i hibit ti it (T bl 1) H i l t f th li d l t

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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 ofPseudomonas 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 weredetected. In the presence ofBacillus licheniformis strains, the fungal mycelium was more

developed on the walls of the test tube, while in presence ofPseudomonasaeruginosa, themycelium had a limited growth, at the surface of the medium. Moreover, in the case of

PseudomonasAlternaria cocultivation, the color of the medium became yellow and

fluorescent under UV illumination, proving the synthesis of fluorescent pigments by thebacterial 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 microscopyIn the early stages of Pseudomonas aeruginosa and Alternaria alternata

cocultivation, the fungal hyphae were rare and isolated from the bacterial colonies (Plate Ie). 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 If) and conidia formation (Plate I j) were observed.

After 5 days from inoculation, in the case of the samples with Pseudomonasaeruginosa, the bacterial cells tended to adhere to the fungal hyphae (Plate I g and h), while

B ill li h if i ll l t d l th h h (Pl t I k d i) Th

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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 predominatesin 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 theliquid 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 vesicleswith fibrilar content are detached from these cell walls (Plate III a and b), as a possibleresponse to the bacterial action, in particular to the bacterial synthesis of lytic enzymes.

The agglomeration of bacterial cells around the fungal hyphae was observed whenthe 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 exhibitingdifferent 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 structuralmodifications observed in the bacterial-fungal interaction were: the contraction of

cytoplasm (Plate IV f and g), secretion of extraprotoplasmic vesicles and degeneration ofmembrane system (Plate IV g), phenomena characteristic for cell plasmolysis. In severe

cases, plasmalemma was disrupted and cytoplasm appeared to lose its structuralorganization (Plate IV f). The presence of bacteria induces a premature senescence and

f th t l i d di i ti f h h t l lti i h t ll t f

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had a higher inhibitory activity, the growth of fungal mycelium being reduced, while the

antagonistic activity ofBacillus licheniformis was more stable in time.Pseudomonas aeruginosa strains produced more diffusible compounds thanBacillus

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 ofBacilluslicheniformis BC98 (Tendulkar et al., 2007).

The release of fluorescent pigments byPseudomonas aeruginosa strains varies withantifungal activity, thus the culture media of P14 and P18 strains emitted a highfluorescence, 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 resultof 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. Thefilamentous 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, thefungal 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 thecontrol samples, this coat of fungal cell wall was not observed, thought in classical

t t t l d i ti f t h h th ll d ith l b

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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. Themelanins can protect fungal cell walls components against degradation by inhibition of lytic

enzymes as chitinases and glucanases. Also, the melanin may provide protection againstfungicide 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, thebacterial 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 inthe sections that present only fungal cells. The main conclusion that can be derived fromthis 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 structuralmodifications at the cell wall level were the apparition of protuberances, release of vesiclesor 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. Drasticmodifications, 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

d t f i id l d t t d i lt ith B ill li h if i t i

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Campbell, R.,1969. An electron microscope study of spore structure and development in Alternaria brassicicola.

J. Gen. Microbiol. 54, 3: 381-392.Campbell, R., 1970. Ultrastructure of an albino strain ofAlternaria brassicicola. Trans. Br. Mycol. Soc. 54: 309-

313.Chrif, M., Sadfi,N., Ouellette, G.B., 2003. Ultrastructure of in vivo interactions of the antagonistic bacteria

Bacillus cereus X16 andB. thuringiensis 55T withFusarium roseum var. sambucinum, the causal agentof potato dry rot. Phytopathol. Mediterr. 42: 41-54.

Compant, S., Duffy, B., Nowak, J., Clment, C., Barka, E.A., 2005. Use of Plant Growth-Promoting Bacteria forBiocontrol of Plant Diseases: Principles, Mechanisms of Action, and Future Prospects. Appl. Environ.Microbiol. 71: 4951-4959.

Cornea, C.P., Ciuc, M., Voaide, C., Constantinescu, F., Keepferberg, S., Voicu, A., tefnescu, M., Bbeanu,N., 2006. Genetic diversity of some bacterial strains with biotechnological importance. Lucrri tiintifice

USAMVB, seria F, Biotehnologii. X: 39-46.

Deora, A., Hashidoko,Y., Islam, M.D.T., Aoyama, Y., Ito, T., Tahara, S., 2006. An antagonistic rhizoplanebacterium Pseudomonas sp. strain EC-S101 physiologically stresses a spinach root rot pathogenAphanomyces cochlioides. J. Gen. Plant Pathol. 72: 57-64.

El-Ghaouth, A., Wilson, C.L., Wisniewski, M., 1998. Ultrastructural and Cytochemical Aspects of the BiologicalControl ofBotrytis cinereaby Candida saitoana in Apple Fruit. Phytopathology. 88: 282-291.

Handelsman, J., Stabb, E.V., 1996. Biocontrol of Soilborne Plant Pathogens. Plant Cell. 8: 1855-1869.Lee, J.Y., Moon, S.S., Hwang, B.K., 2003. Isolation and Antifungal and Antioomycete Activities of Aerugine

Produced byPseudomonas fluorescens Strain MM-B16. Appl. Environ. Microbiol. 69: 2023-2031.Lee, Y-S., Kim, J., Shin, S-C., Lee, S-G., ParkI-K., 2008. Antifungal activity of Myrtaceae essential oils and their

components against three phytopathogenic fungi. Flavour and Fragrance Journal. 23: 23-28.Mascorro, J.A., Bozzola, J.J., 2007. Processing Biological Tissues for Ultrastructural Study, in: Kuo, J. (Ed.).

Electron Microscopy. Methods and Protocols. Humana Press: 19-35.

Mateescu, R., Cornea, C.P., Grebenian, I., Drago, ., Sirean, O., Jurcoane, ., Cmpeanu G., 2003. Theimprovement of antifungal potential of some bacilli strains by electrotransformation. Roum. Biotechnol.Lett. 8, 4: 1367-1374.

Mateescu, R., Cornea, C.P.,Grebenian, I., Sirean, O., 2005. Improvement by mutagenesis and electroporation of

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Table 1. Inhibitory activity ofBacillus licheniformis andPseudomonas aeruginosa strains against different fungal phytopathogens

Phytopathogens Bacterial

strains

Inhibitory activity (%)

2 days 3 days 4 days 5 days 6 days 7 days 8 days 9 days 10 days 13 days 14 days

Pythium

debaryanum

P7 ns 26.66 4.7 31.54 1.8 33.92 3.3 18.58 1.6 - - - - - -

P14 ns 32.77 5.8 41.83 4.1 44.54 3.1 42.57 2.7 37.85 8.2 29.99 8.2 ns ns ns ns

P18 ns 33.33 4.7 41.61 0.9 43.36 3.3 38.64 3.3 ns ns ns ns ns ns

B40 ns 27.22 0.9 39.14 3.0 40.60 0.6 38.64 2.0 37.85 1.3 37.06 1.8 37.46 1.7 37.46 37.06 37.06 1.8

mB40 ns 28.33 1.6 40.04 0.7 41.00 1.1 39.42 1.3 39.82 1.1 39.03 1.8 37.85 1.3 37.85 37.85 37.85 1.3

tB40 - ns - ns ns ns ns - - - -

Fusarium

oxysporum

P7 40.74 ns ns ns 29.74 13.8 25.33 26.05 28.45 8.6 27.27 27.02 26.25 12.0

P14 ns ns ns ns ns ns ns 20.32 22.22 26.12 25.83 10.4

P18 ns ns ns ns ns 14.00 4.0 17.32 4.3 16.53 4.0 19.19 20.72 23.33 10.1

B40 - - - ns ns ns ns ns 23.23 ns 29.58 10.1

mB40 - - - ns 7.52 2.1 ns 18.48 1.0 21.40 1.8 27.27 36.03 41.25 0.0 (b)

tB40 ns ns ns ns ns ns ns ns ns ns 27.5 10.8

Botrytis cinerea

P7 ns ns ns - - - - - - - -

P14 ns 25.68 6.8 20.34 1.4 ns ns - - - - - -

P18 ns 32.24 1.8 20.34 1.4 ns - - - - - - -

B40 ns 32.24 2.5 46.32 1.9 46.58 1.4 46.58 1.4 45.72 1.9 46.15 2.2 45.29 2.6 45.29 45.72 45.72 2.9mB40 ns 28.96 3.7 41.99 3.9 41.45 4.5 41.45 4.5 40.17 4.1 41.45 4.5 41.45 4.5 41.45 43.58 43.58 4.4

tB40 ns ns ns ns - - - - - - -

Alternaria

alternata

A1 strain

P7 - ns 32.33 3.4 36.30 32.28 8.7 ns ns ns ns ns ns

P14 - ns 39.30 1.7 44.79 ns 43.15 2.8 41.48 3.8 39.81 4.3 37.30 35.63 35.63 3.8

P18 - ns 36.31 1.7 40.55 38.97 0.7 36.05 3.7 34.37 5.0 33.12 32.70 28.94 28.94 3.8

B40 - 31.29 2.3 50.24 2.2 57.53 1.9 57.78 1.4 49.84 54.44 0.7 54.44 0.7 51.93 50.26 50.26 0.7

mB40 - ns 44.77 6.3 52.22 4.5 51.09 5.3 47.96 4.4 48.58 5.3 48.58 5.3 47.33 47.96 47.96 4.4

tB40 - ns ns 34.18 4.0 29.78 6.6 24.76 2.5 25.60 3.8 21.42 3.8 20.58 ns ns

Alternaria

alternata

A100 strain

P7 - ns ns ns 21.77 6.4 ns - - - - -

P14 - - ns ns 31.11 1.7 27.18 1.7 25.21 1.7 23.24 2.9 23.24 20.29 20.29 2.9

P18 - - ns ns 30.62 4.4 30.13 6.8 19.80 2.2 16.35 4.5 14.39 ns ns

B40 - - ns ns 43.91 2.0 43.91 2.0 43.17 1.0 43.17 1.0 43.17 43.17 43.17 1.0

mB40 - ns 26.85 8.2 36.12 10.2 45.38 8.9 43.91 5.1 41.94 3.0 42.43 3.9 42.43 41.45 41.45 3.7

tB40 - - - - ns - - - - - -

- no activity; ns - no significant; data- significantly different than control (p0.05).

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