CHARACTERIZATION OF BACTERIAL

ENDOPHYTES FROM IN VITRO

RASPBERRY SHOOT CULTURES
Teresa Orlikowska, Marta Zawadzka
Research Institute of Horticulturae, Skierniewice, Poland

Isolation of bacteria contaminating raspberry

shoot cultures derived from four different in vitro
laboratories
All contaminations visible by naked eye and in binocular water-halos or
colour rings around shoots, and leakages in the medium were sampled
and inoculated on 6 media: NA, KING B, K, 523, R2A and MS with 200
mg/l milk albumine (typical initiation medium). Also samples from
suspected cultures were taken from the base of shoots or from the
medium beneath.
35 bacterial isolates were separated and identified by microbiological
tests and DNA sequencing, as Curtobacterium flaccumfaciens, Klebsiella
spp., Methylobacterium extorquens, M. lusitanum, Methylobacterium spp.,
Mycobacterium spp., Paenibacillus spp., Pseudomonas putida,
Staphylococcus pasteuri, and Stenotrophomonas spp.

The useability of media for bacteria isolation

Only one Staphylococcus pasteuri was isolated from a necrotic shoot.
All the rest contaminated cultures did not display symptoms of
pathogenesis.
Among 6 tested media the 523 medium (Viss et al., 1991) promoted
growth of all bacteria contaminating raspberry cultures better than other
media. Medium K, recommended for Methylobacteria (Ivanova et al.,
2005) was much less favourable for the isolation of Methylobacteria
from contaminated raspberry cultures.

The morphology of raspberry

microshoots contaminated with
Curtobacterium flaccumfaciens

Methylobacterium sp. in
common elder culture

The influence of three isolated bacteria on plant

tissue cultures material & methods
Paenibacillus sp., Curtobacterium flaccumfaciens and Methylobacterium
lusitanum strains isolated from contaminated raspberry cultures were
choosen for the experiment basing on visual observations of
contaminated cultures.
Four plant genotypes were included: chrysanthemum Ludo, gerbera
Kormoran, hosta Blue Cadet and rosa White Gem.

The shoots for the experiment were collected from stock cultures free of
cultivable bacteria. The stocks originated from individual microshoots
screened in two subsequent subcultures for the presence of cultivable
bacteria by incubation of the bottom parts on two bacterial media NA
and 523.

Prepairing of explants for experiments

Shoots were inoculated with bacteria at rooting in the perlite saturated with
a liquid MS auxin-containing medium. The volumes of 100 l of 24h
bacterial cultures were injected around shoots.
After 4 weeks, the bottom parts with roots were cut-out and the shoot tips
were transferred to the multiplication media. A visual observation of bacteria
presence in the forms of leakages or halos was accompanied by molecular
confirmations of the bacterial DNA.
For multiplication and rooting the media typical for each plant species were
used.

Chrysanthemum
c

Rosa

a
a
a

a
ab a b

Gerbera

b a

Hosta

M. lusitanum

C. flaccumfaciens

b a

Paenibacillus spp.

control

Length of shoots (cm)

M. lusitanum

C. flaccumfaciens

Paenibacillu spp.

control

M. lusitanum

C. flaccumfaciens

Paenibacillus spp.

control

M. lusitanum

C. flaccumfaciens

Paenibacillus spp.

control

The influence of bacteria on shoot multiplication

data from 30 microshoots per treatment
No. of shoots

c
a
b

a
c
c

a
c

a
b

The influence of bacteria on rose shoot

multiplication

Control

M. lusitanum

C. flaccumfaciens

Paenibacillus sp.

Chrysanthemum
Rosa
b

a
a

a
b
c

Gerbera

a
a

Hosta

M. lusitanum

a
b

C. flaccumfaciens

1
c

Paenibacillus spp.

control

No. of roots

M. lusitanum

C. flaccumfaciens

Paenibacillus spp.

2
a

control

M. lusitanum

C. flaccumfaciens

Paenibacillus spp.

control

M. lusitanum

C. flaccumfaciens

Paenibacllus spp.

control

The influence of bacteria on rooting of microshoots

Length of roots (cm)
c

b
c

c
b

b
a

The influence of bacteria on rooting of rose

microshoots

Control

M. lusitanum

C. flaccumfaciens

Paenibacillus sp.

The influence of bacteria on rooting of microshoots

in vitro versus in vivo
Shoots were inoculated with bacteria at rooting in the perlite saturated
with a liquid MS auxin-containing medium. The volumes of 100 l of 24h
bacterial cultures were injected around shoots.
After 4 weeks, the bottom parts with roots were cut-out and the shoot tips
were transferred to the multiplication media. A visual observation of
bacteria presence in the forms of leakages or halos was accompanied by
molecular confirmations of the bacterial DNA.
For multiplication and rooting the media typical for each plant species
were used.

There were 30 microshoots per treatment.

The influence of bacteria on the rooting of gerbera

microshoots inoculated by Methylobacterium lusitanum
IAA

Methylobacterium

IAA + Methylobacterium

in vitro

in vivo

The influence of bacteria on the rooting of hosta microshoots inoculated by Methylobacterium lusitanum
NAA

Methylobacterium

NAA + Methylobacterium

in vitro

in vivo

The influence of bacteria on the rooting of chrysanthemum microshoots inoculated by Paenibacillus sp.
IAA

Paenibacillus

IAA + Paenibacillus

in vitro

in vivo

The influence of bacteria on the rooting of rose microshoots inoculated by Curtobacterium flaccumfaciens
IBA

Curtobacterium

IBA + Curtobacterium

in vitro

in vivo

The influence of bacteria on rooting in vitro

versus
ex vitro
in vitro

in vivo

The influence of bacteria on the acclimatization

All microshoots produced roots both in vitro and in vivo.
A transfer to the greehouse survived from 50 to 100 %
microplants rooted in vitro, depending on the plant genotype.

All microshoots rooted in the greenhouse survived.

During the first 4 weeks after planting the microplants rooted in
vitro were lower and looked weaker but after that time the
growth was equalized and 6 weeks later no significant
differences were observed.

Conclusions
1. Cultivable bacteria are common in raspberry tissue cultures.
2. The universal medium for revealing the presence of bacteria
contaminating raspberry cultures was 523.
3. Three selected bacterial strains belonging to Paenibacillus sp.,
Curtobacterium flaccumfaciens and Methylobacterium lusitanum
can stimulate the shoot and root growth in vitro but a kind of
specialisation between the bacteria/plant genotypes was
observed.
4. The most interesting practical result is that bacteria can improve
the root growth and enrich the root architecture at rooting in a
common substrate in the greenhouse in comparison to the rooting
in vitro.
5. The direct rooting in vivo is more effective than in vitro due to
100% survival of microshoots.

Bacteria

Isolated from

Action

Mechanism

Authors

Azospirillum
brasiliense Cd i Sp7

Rhizosphere

Facilitates acclimatisation of
Photinia

More rootlets

Larraburu et.al. 2007,

2010

Azospirillum
brasiliense Az39

Rhizosphere

Increases root system under

salt stress

Produces
cadaverine
(polyamin)

Cassan et al., 2009

Azospirillum
brasiliense Sp245

Rhizosphere

Enriches root system, protects

Prunus before
Rhizoctonia spp

Azospirillum
brasiliense Sp245

Rhizosphere

Facilitates acclimatisation of
apple and cherry rootstocks

Azotobacter
chroococcum

Rhizosphere

Increases more side shoots of

Simmondsia

Azotobacter
chroococcum

Microorg.
collection.

Facilitates acclimatisation of
Morus, increases NaCl
tolerance

Facilitates P and
N absorption

Kashyap i Sharma,
2006

Azorrhizobium
caulinodans

Leguminose
shoots

Increases protein level in rice

calli, stimulates adventitious
shoots regeneration

Assimilation of air
N

Senthikumar et al.,
2008

Russo et al. 2008

Increases root
number through
IAA

Vettori et al., 2010

Andressen et al.,
2009

Bacteria

Isolated from

Action

Mechanism

Authors

Bacillus 7 strains

Strawberry
meristems in vitro

Stimulates root growth

Produces IAA

Dias et al., 2009

Bacillus subtilis

Bacillus circulans

From
pelargonium
seedlings in vitro

Bacillus spp. (INR7,

T4, IN 637b)

Increases resistance of potato

microplants for Rhizoctonia
solani

Cassels et al.,
2003

Stimulates somatic
embryogenesis of
pelargonium

Murthy et al.,
1999

Fastests growth of banana

microplants

Increases amounts of
NPK in plant tissues

Jaizme-Vega et
al., 2004

Burkholderia
phytofirmans PsJN

Onion roots

Facilitates acclimatisation of
several plant species

ACC deaminase
activity, activity of
compunds
restraining quorumsesing

Sessits et al.,
2005

Burkholderia
phytofirmans PsJN

Onion roots

Inreases resistance of grape

to gray molds and to low
temperatures

Increases activity of
stress genes of
plants

Theocharis et al.,
2012

Bacteria

Isolated from

Action

Mechanism

Authors

Curtobacterium
citreum 3
morphotypes

Covert contamination
from
Chrysanthemum
tissue culture

Availables chrysanthemum
micropropagation without
growth regulators in medium

Produces
cytokinin
(5-20 uM)

Panicker et al.,
2007

Enterobacter spp.
SC11, 12, 13, 20

Sugar cane tissues

Stimulates fresh weight of

sugar cane microshoots

Produces IAA
assimilates N

Mirza et al.,
2001

Halomonas
desiderata RE1

Rhizosphere of plants
tolerating hogh salt
level

Stimulates shoot and callus

growth, induces side shoots of
Brassica oleracea

Produces IAA (21

ug/ml of bacterial
supernatant

Ali Hasnain,
2007

Methylobacterium
extorquens

Strawberry callus

Stimulates callus growth,

produces scent compounds

produces2 furans,
possesses
oxydative
processes

Zabetakis, 1997

Methylobacteriu sp.
D10, Methylophilus
glucoseoxydans

Soil and rhizosphere

of rice

Stimulates callus growth and

growth of regenerated shoots

Produces ipt and

IAA

Kalyaeva et al.,
2003

Methylovorus mays
BKM B-2221

Rhizosphere

Increases resistance of rape

for fungal pathogens, facilitates
acclimatisation

Zakharchenko et
al., 2012

Bacteria

Isolated from

Action

Pseudomonas sp.
PsJN

Onion roots

More roots, longer shoots, more

mature tissues, more effective
stomatal closiure, mor lignin of
potato

Frommel et al.,
1991

As above

As above

Stimulates shoot growth, number

of nodes, weight shoots and roots,
increases resistance to gray mould
of grape

Ait Barka et al.,

2001

As above

As above

Tomato plants more resistant to

Verticillum daliae

Sharma and
Nowak, 1998

Pseudomonas
aureofaciens BS
1393

Bank of microorganisms

Facilitates growth and

acclimatisaton of potato and
strawberry microplants, increases
resistance to pathogenic fungi

Zakharchenko et
al., 2011

Pseudomonas
stutzeri P3

Echinacea tissue
cultures

Pseudomonas
fluorescens L 26-1 i
P. putida

Mechanism

IAA production
Faciilitates acclimatisation of
Primula

Authors

Lata et al., 2006

Digal et al., 1987

Bacteria

Isolated from

Action

Mechanism

Authors

Paenibacillus sp. P22

Woody plants tissue

cultures

Stimulates growth of root

system

Changes in 11
metabolits, N
assimilation

Scherling et al.,
2009

Rhodococcus
fascians

Echinacea tissue
cultures

Produces different
plant growth
regulators, including
rodestrin

Vareecke et al.,
2000

Sphingopyxis

Meristem cultures of
strawberry

Facilitates acclimatisation
and shoot growth

IAA production,
utilisation of P

Dias et al., 2009

Rhizobium sp.

Noduls of black
accacia

Facilitates acclimatisation

N asimilation

Balla et al., 1998