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s. II a. Biologie vegetal, 2012, 58, 2: 5-18 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 Growth-
Promoting 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
 Mitoi, M.E. et al., 2012/ An. Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 58, 2: 5-18

(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 2-
3 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

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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.

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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 bacteria-
pseudomonads 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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 Mitoi, M.E. et al., 2012/ An. Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 58, 2: 5-18

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: 5-18

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: 5-18

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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 Mitoi, M.E. et al., 2012/ An. Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 58, 2: 5-18

Table 1. Inhibitory activity of Bacillus licheniformis and Pseudomonas aeruginosa strains against different fungal phytopathogens

Phytopathogens Bacterial Inhibitory activity (%)

strains 2 days 3 days 4 days 5 days 6 days 7 days 8 days 9 days 10 days 13 days 14 days
P7 ns 26.66 4.7 31.54 1.8 33.92 3.3 18.58 1.6 - - - - - -
Pythium 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(a) 3.3 38.64 3.3 ns ns ns ns ns ns
debaryanum B40 ns 27.22 0.9 39.14 3.0 40.60(b) 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(b) 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 - (b)
ns ns ns ns - 0.9
- 1.8
- -
(b) 1.1 1.3
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
Fusarium P14 ns ns ns ns ns ns ns 20.32 22.22 26.12 25.83 10.4
P18 9.8
ns ns ns ns ns 11.5
14.00 11.8
4.0 17.32 4.3 16.53 4.0 6.5
19.19 9.3
20.72 (ab)
23.33 10.1
oxysporum B40 - - - ns ns ns ns 10.1
ns 7.9
23.23 7.8
ns (ab)
29.58 10.1
mB40 - - - ns 7.52 2.1 ns 18.48 1.0 21.40 1.8 1.8
27.27 6.8
36.03 41.25 (a)
0.0 (b)
tB40 ns ns ns ns ns ns ns ns 8.4
ns ns 27.5(ab)
10.8
1.2 1.5
P7 ns ns ns - - - - - - - -
P14 ns 25.68 6.8 20.34 1.4 ns ns - - - - - (ab)
-
Botrytis cinerea P18 ns 32.24 1.8 20.34 1.4 ns - - - - - - -
B40 ns (a) 2.5
32.24 (a) 1.9
46.32 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 2.9
mB40 ns (a) 3.7
28.96 (a) 3.9
41.99 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 (a)
ns (b)
ns (b)
ns - - - - 2.1
- 2.9
- -
(a) (b) (b) 3.6 4.4
P7 - ns 32.33 3.4 36.30 32.28 8.7 ns ns ns ns ns ns
Alternaria
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 2.2(a)
40.55 38.97 0.7 36.05 3.7 34.37 5.0 33.12 32.70 28.94 28.94 3.8
alternata
B40 - 31.29 2.3 50.24 2.2 5.3(a)
57.53 1.9 57.78 1.4 49.84 54.44 0.7 54.44 0.7 3.6
51.93 3.8
50.26 50.26 0.7
A1 strain mB40 - ns 44.77 6.3 1.4(a)
52.22 4.5 51.09 5.3 47.96 4.4 48.58 5.3 5.2 5.3
48.58 4.1
47.33 3.8
47.96 47.96 4.4
tB40 - ns ns 34.18(b) 4.0 29.78 6.6 24.76 16.8 2.5 25.60 3.8 21.42 3.8 0.5
20.58 0.7
ns ns
(b) 2.5 4.4
Alternaria P7 - ns ns ns 21.77 6.4 ns - - - - -
P14 - - ns (a)
ns 31.11 1.7 27.18 1.7 25.21 1.7 23.24 2.9 2.3
23.24 20.29 20.29 2.9
alternata P18 - - ns ns 30.62(b) 4.4 30.13 6.8 19.80 2.2 16.35 4.5 14.39 ns ns
B40 - - ns ns 43.91(b) 2.0 43.91 2.0 43.17 1.0 43.17 1.0 2.4
43.17 2.9
43.17 43.17 1.0
A100 strain (b) 8.9 43.91 5.1 41.94 3.0 2.4
mB40 - ns 26.85 8.2 36.12 10.2 45.38 42.43 3.9 42.43 41.45 41.45 3.7
tB40 - - - - (b)
ns - - - 0.7
- 1.0
- -
(b) 3.1 3.7
- 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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