Annals of Agrarian Science 15 (2017) 213e216
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Annals of Agrarian Science
journal homepage: http://www.journals.elsevier.com/annals-of-agrarianscience
Bacterial diseases of tomato plants in terms of open and covered
growing of Ukraine
J.V. Kolomiets a, *, I.P. Grygoryuk a, L.M. Butsenko b
a
b
National University of Life and Environmental Sciences of Ukraine, 15, Geroev Oborony Str., Kyiv, 03041, Ukraine
Zabolotny Institute of Microbiology and Virology of the NAS of Ukraine, 154, Acad. Zabolotny Str., Kyiv, 03680, Ukraine
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 June 2016
Accepted 22 February 2017
Available online 10 May 2017
It was established that the main causes of mass diseases of tomato in covered ground in Ukraine are
agents of bacterial black spotting, bacterial speck and in open ground are agent of bacterial cancer of
tomato plants. Typical symptoms of diseases are wilting and die-off of young plants, blackening of fiber
vascular bundles, black spotting of leaves and fruits, and fruit stem rot. It was studied morphological and
cultural, as well as physiological and biochemical properties of the selected strains of the agents of tomato bacterial diseases. We recommended biological preparations Phytocide and Phytohelp based on the
bacteria Bacillus subtilis, to restrict the development of the agents of bacterial black spotting Xanthomonas vesicatoria and bacterial cancer Clavibacter michiganensis subsp. michiganensis.
© 2017 Agricultural University of Georgia. Production and hosting by Elsevier B.V. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords:
Tomato
Bacterial speck
Crop
Agrocenoses
Pathogen
Introduction
In tomato plants it can be found a number of bacterial diseases
affecting various organs: stem centrum necrosis, bacterial leaf
spotting, fruit mottling (spotting), fruit top rot, watery rot of stems
and fruits, bacterial wilt (brown rot) of stems, root cancer.
The frequency and intensivity of disease outbreaks in major
tomato growing areas of Odesa, Zaporizhia, Mykolaiv, Dnipropetrovsk, and Kherson regions of Ukraine varied from year to year and
depend on weather and climatic factors, as well as different degrees
of compliance with environmental requirements of a certain agent
[1].
In Ukraine, in terms of open and covered growing the most
common disease is bacterial black spotting. The agent Xanthomonas
vesicatoria affects aboveground plant parts, resulting in reduced
harvest down to 10e20%; if the lesion is severe, the fruits are not
formed, and the affected fruits lose their quality and their flavor is
reduced. The high average temperature promotes to mass spread
and development of the disease. At the end of the vegetation period
the spread and the development of bacterial black spotting reaches
95 and 40% respectively, the degree of fruit destruction e 1,7%.
Major crop losses are observed in the places of permanent growing
* Corresponding author.
E-mail address: julyja@i.ua (J.V. Kolomiets).
Peer review under responsibility of Journal Annals of Agrarian Science.
of tomatoes and varieties of the nightshade family, especially of
potato [2,3].
In Zaporizhia and Dnipropetrovsk regions of Ukraine it is
observed the development of bacterial speck (the agent is Pseudomonas syringae pv. tomato), which is developed due to high humidity and low night temperatures (12e15 C). Lesions are
observed in all above-ground plant organs, especially in fruits [3,4].
According to phytosanitary state of agrocenoses in Ukraine and
recommendations for protecting tomato plants, bacterial speck
manifests itself in the first decade of July, in August the infestation
is 1e45%, maximum 68, with the development of the disease e
0,3e6, infestation of fruits e 0,5e12% [5].
During the last decade 2005e2015 at the Ukrainian farms,
especially in greenhouses, it was repeatedly observed mass dyingoff of the tomato plants which was accompanied by withering the
leaves and the whole plant [6]. The main cause of bacterial wilt of
tomato in covered and open ground is Clavibacter michiganensis
subsp. michiganensis, the agent of tomato bacterial cancer. It is
especially dangerous in the covered ground, causing massive
destruction of plants during the fruiting period. The pathogen
mainly penetrates into a plant through the mechanical damages
and roots [7].
To protect plants from infectious organisms are used pesticides
in agriculture. In the second edition of the official “List of pesticides
and agrochemicals permitted for use in Ukraine for 2010”, there is
no information on the drugs recommended for bacterial lesions of
tomato plants [8]. According to the obtained data, fungicides, which
http://dx.doi.org/10.1016/j.aasci.2017.05.010
1512-1887/© 2017 Agricultural University of Georgia. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
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214
J.V. Kolomiets et al. / Annals of Agrarian Science 15 (2017) 213e216
include mankozeb or copper hydroxide, significantly inhibit the
growth of bacterial agents and the development of cancer and
bacterial black spotting [9].
Recently, in Ukraine it was renewed the interest in biological
methods of plant protection, which is based on the use of microorganisms or their metabolic products to inhibit the development
of pathogens. Biologicals based on live bacterial cultures are characterized by low toxicity and broad spectrum of activity against
plants and pathogens. The use of combined biologicals that include
properties of bio-fertilizers, fungicides, and insecticides, makes it
possible to solve a large number of problems of biological protection of plants and improve the quality of final products (vegetables,
fruits), as well as soil fertility [10].
Due to the heavy infestation of tomato with bacteriosis, the aim
of our study was to study and identify agents of bacterial diseases of
tomato in terms of open and covered ground and to search for
protection remedies.
Objectives and methods
Tomatoes with symptoms of bacterial lesions were taken in
different periods of growth from the farms of Dnipropetrovsk and
Zaporizhia regions. The study was conducted by standard microbiological and phytopathological methods [11]. Pieces of infected
plant tissues were successively washed with non-sterile water and
then with sterile water, homogenized in a sterile mortar and plated
on potato agar strips in Petri dishes. Colonies of grown bacteria
were selected for further study of their pathogenic, morphological,
biochemical, and physiological properties [11]. Pathogenic characteristics of isolates were studied in fruits and vegetative plants of
tomatoes using suspensions of bacteria cells with titer 1 108 CFU/
cm3. Fruits were infected through injection with a drop of suspension, stems e wounding with Pasteur pipette, and the capillary
of which is filled with a suspension of cells was left in the tissue.
In experiments they were used collection strains of phytopathogenic bacteria Xanthomonas vesicatoria (Doidge 1920) Vauterin et al., 1995 strain 9098 from the collection of phytopathogenic
bacteria department of Zabolotny Institute of Microbiology and
Virology of the NAS of Ukraine; Clavibacter michiganensis subsp.
michiganensis (Smith 1910) Davis et al., 1984 strain P8 and Pseudomonas syringae pv. tomato (Okabe 1933) Young et al., 1978 strain
Dappg-4213 obtained from the Institute of pesticides and plant
protection in Serbia.
Biologicals were used in concentrations recommended by the
manufacturer, including Phytohelp e concentrated mixture of
natural bacteria Bacillus subtilis (4х109 CFU/cm3), micro- and macroelements, biologically active products of microorganisms (BTUcenter, Ukraine) [12], Phytozid e living cells and spores of natural
endophyte bacterium Bacillus subtilis (1х109 e 4х109 CFU/cm3),
their active metabolites (BTU-center, Ukraine) [12], Ekstrasol e
strain of rhizospheric, nitrogen-fixing bacteria Bacillus subtilis Ch13 (1х108 CFU/cm3) (LLC “Bisolbi-Inter”, Russia) [13], Planriz e
culture fluid rhizospheric bacteria Pseudomonas fluorescens AP-33
(5 109 CFU/cm3) and biologically active substances (BAS) (OOO
“Center Biotekhnika”, Ukraine) [14], Haupsyn e culture liquid of
two strains of ground bacteria Pseudomonas aureofaciens
(4,5х109 CFU/cm3) and BAS (LLC “Biotechnica center”, Ukraine) [14],
Azotofit e the cells of natural nitrogen-fixing bacteria Azotobacter
chroococcum (1х109 CFU/cm3), macro- and microelements, BAS of
bacteria (BTU-center, Ukraine) [12].
The effect of biologics on bacteria was studied by method of
diffusion in agar [15]. In Petri dishes into holes made by sterile cork
drill in the middle of congealed potato agar, with the help of sterile
removable spouts it was added biological in the recommended by
manufacturer concentrations. Then it was radially plated one-day
old bacteria with titer 1 109 CFU/cm3. The dishes were incubated for 48 h at a temperature of 28 ± 1 C. The antibacterial effect
of the substance was set due to the diameter of the zone with
absence of bacterial growth.
Statistical analysis of the results was performed using application soft ware package STATISTICA v.6.0.
Results and analysis
In the open ground the most often e were observed affected
plants with symptoms that were identified the most clearly on the
leaves and fruits. On the leaves were observed brown dots
(1e2 mm), which were surrounded by a chlorotic areols. Single dots
were mostly observed at the lamina edge and near veins. In mass
infection almost the entire leaf surface acquired chlorotic coloration. Sometimes the affected area grew, forming large dots (Fig. 1).
Leaves acquired brown coloration, and then blackened, withered,
but did not drop off. Plants gradually died off.
From the leaves infection was passed to the fruits. They
observed two types of necrosis: some of them were upraised above
the surface, and the others e flat (Fig. 2). Compacted dark brown
spots with torn edges, resembling a scab formed watery zones
around themselves (Fig. 2, A). We selected fruits with upraised dark
spots with a diameter of 1e5 mm (Fig. 2 B), and small dark brown
slightly convex dots (Fig. 2C, D). The tissue under the spots in
mature fruits began to rot.
In the covered ground leaves of most affected plants were
flaccid, the leaves rolled, acquired corrugated look, became brown
and withered. The cause of wilting and dying of tomato plants in
greenhouses were affected stem vessels found in the transversal
and vertical sections (Fig. 3). Under the influence of bacteria, the
vessels first acquired yellowish or light brown color, then increasingly darkened and eventually became dark brown. Bacteria
infected separate bundles and sometimes all the stem vessels.
Depending on the number of affected vessels it was observed
unilateral wilting of leaves or wilting of the entire plant. The plant
died not necessarily due to the destruction of all vessels, but also
due to affection of most of them. This indicates that bacteria not
only mechanically sealed the vessels, disturbing the water regime,
but also the fact that the dying of the plants was also due to the
toxins of bacteria. The number and color of the affected vessels can
determine the location and direction of the infection. According to
the literature, in greenhouses Kiev, Kharkiv and Kherson regions of
Ukraine in the affected vessels was often the agent of bacterial
canker with soft rot pathogen Pectobacterium carotovorum.
During studying tomatoes in open and covered ground according to the described symptoms of infections, the disease is
attributed to one of three the most harmful in Ukraine (Table 1).
Under laboratory conditions as a result of bacteriological analysis of the selected leaves and fruits with signs of affection we
allocated three morphological types of bacteria isolates.
To the first type we belong round, convex, yellow with smooth
edges slimy colonies characteristic for X. vesicatoria. Studying the
biological properties of isolates, compared to the collection strains
and to the data of bacteria determiners, showed that the separated
bacteria isolates are Gram-negative, oxidase-negative, catalasepositive, thin rods 0,6 1,0e1,5 mm, inactive, with lack of nitrate
recovery, indole formation, producing acid from glucose, mannose,
galactose, hydrolyzed gelatine slowly, didn't use lactose, rhamnose,
sorbitol, formed transculent colonies on the media with
carbohydrates.
Isolates of the second type are gray and white, round, slightly
upraised with smooth shiny surface colonies, rods 0,6 1,5e3 mm,
mobile, moving forward, gram-negative, while growing in beefextract broth it was observed turbidity with a film on the surface,
J.V. Kolomiets et al. / Annals of Agrarian Science 15 (2017) 213e216
215
Fig. 1. Symptoms of bacterial diseases on leaf plates of tomato plants: A e dark spots with a diameter of 3e4 mm (the agent of X. vesicatoria), B e damage, surrounded by chlorotic
zone (the agent of P. syringae pv. tomato), C e necrotic areas on the lamina edge (the agent of C. michiganensis subsp. michiganensis).
Fig. 2. Symptoms of bacterial diseases on immature fruits of tomato plants: A e spots of watery edges and light center (the agent of C. michiganensis subsp. michiganensis), B e spots
of convex form (the agent of X. vesicatoria), C e small convex dots 1e5 mm and D e 1e3 mm (the agent of P. syringae pv. tomato).
Fig. 3. Symptoms of Bacterial cancer on stem vessels of tomato plants.
catalase-positive, utilizing citrate, didn't form indole, produced acid
from glucose, sucrose, inositol, did not assimilate lactose, maltose,
arabinose, xylose, valine, tryptophan and threonine, caused green
fluorescence in beef-extract broth. The results of comparative
analysis show that the isolates according to the totality of biological
properties are identical to species Pseudomonas syringae pv. tomato.
To identify the bacteria Pseudomonas syringae it is often used LOPAT
test. According to these properties the separated phytopathogenic
bacteria are identical to Pseudomonas syringae pv. tomato, as they
produce levan, are oxidase-negative, do not cause maceration of
plant tissues, do not have arginine dihydrolase, and cause hypersensitivity reactions development in tobacco leaves.
Isolates of the third type are round convex colonies with a
smooth edge, opaque yellow, drop-shaped, non-slimy characteristic
to C. michiganensis subsp. michiganensis. Rods 0,3 1,0e1,4 mm,
inactive, Gram-positive, did not form levan from sucrose, oxidase-
Table 1
The bacterial diseases of tomato plants in Zaporizhia and Dnipropetrovsk regions of Ukraine.
The disease
The agent
The terms of growing
Bacterial cancer
Bacterial speck
Bacterial black spotting
Clavibacter michiganensis subsp. michiganensis
Pseudomonas syringae pv. tomato
Xanthomonas vesicatoria
open and covered growing
open growing
open growing
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J.V. Kolomiets et al. / Annals of Agrarian Science 15 (2017) 213e216
Table 2
The antibacterial activity of the biologicals against the agents of bacterial cancer,
bacterial black spotting and bacterial speck of tomato plants.
The biological
The no growth zone diameter (mm) of strains around the
hole with biologicals, М±m
Clavibacter
michiganensis subsp.
michiganensis
Phytocide
Azotofit
Phytohelp
Planriz
Haupsyn
Ekstrasol
73
78
79
21
28
32
±
±
±
±
±
±
2,0
2,0
1,0
1,0
3,0
3,0
Pseudomonas
syringae pv.
tomato
16
0
17
17
15
23
± 1,0
±
±
±
±
2,0
2,0
1,0
3,0
Xanthomonas
vesicatoria
80
40
80
14
20
40
±
±
±
±
±
±
4,0
3,0
2,0
1,0
2,0
2,0
negative, catalase-positive, utilized citrate, did not hydrolyze
casein, starch, produced acid from glucose and mannose, galactose,
variable e at arabinose, maltose, lactose, raffinose. It is also known
that the phenotypic characteristics of strains C. michiganensis
subsp. michiganensis not depend on the allocation space, a body of
the host plant and the storage period in vitro.
The next stage of the study was to compare the antibacterial
activity of the investigated biologicals. Biologicals Phytohelp, Phytocide and Ekstrasol, based on the bacteria Bacillus subtilis, showed
different antibacterial activity to phytopathogens, due to the peculiarities of the used strains, cells titer and concentration of biologically active products of microorganisms. Biologicals Phytohelp
and Phytocide showed high antibacterial activity against the agents
of bacterial cancer C. michiganensis subsp. michiganensis and bacterial black spotting X. vesicatoria, and the no growth zone diameter
ranged from 73 to 80 mm (Table 2). Under these conditions no
growth zone diameter of the studied strains C. michiganensis subsp.
michiganensis and X. vesicatoria for microbiological preparation
Ekstrasol did not exceed 40 mm. Among these preparations more
active against bacterial speck of tomatoes P. syringae pv. tomato was
microbial preparation Ekstrasol no growth zone diameter of which
was 23 ± 3,0 mm.
Bacilli antagonistic activity against the phytopathogens associates with the synthesis of antibiotics, toxins, volatile organic
compounds, phytohormones, and other exometabolites of different
chemical nature. A significant place in manifestation of antagonism
takes a lytic enzyme complex of bacteria of the Bacillus genus,
which is able to destroy certain connections in the structure of cell
walls peptidoglycan of gram-positive and gram-negative bacteria.
In our study we examined the antibacterial activity of biologicals Azotofit based on nitrogen-fixing bacteria, and Planriz and
Haupsyn based on plant growth promoting bacteria (plant growthpromoting rhizobacteria e PGPR-bacteria). Application of PGPRbacteria is one of biological methods of increasing yields of cultivated plants. PGPR-bacteria positively affect plant growth and
development due to the ability to fix atmospheric molecular nitrogen, to synthesize substances of hormonal nature, improve
water and mineral nutrition of plants, and inhibit the development
of pathogens, due to the synthesis of substances of bactericidal and
fungicidal action.
Biological Azotofit, based on cells of nitrogen-fixing bacteria
Azotobacter chroococcum, showed high antibacterial activity against
the agent of bacterial cancer C. michiganensis subsp. michiganensis,
no growth zone diameter was 78 ± 2,0 mm. This preparation was
middle-active against the agent of bacterial black spotting
X. vesicatoria and showed no activity against the agent of bacterial
speck of tomato P. syringae pv. tomato.
Biologicals Planriz and Haupsyn based on Pseudomonas bacteria
were middle-active against the strains of C. michiganensis subsp.
michiganensis and subactive against the strains of P. syringae pv.
tomato and X. vesicatoria. Antagonist impact of PGPR Pseudomonas
on phytopathogens occurs both through the synthesis of siderophore, antibiotics and other secondary metabolites, and the
simple competition between pseudomonas and phytopathogens
for the sources of nitrogen and carbon supply.
Conclusion
As a result of the research it was found that tomato diseases in
the selected farms of Dnipropetrovsk and Zaporizhia regions of
Ukraine are caused by bacteria, namely by the agents of bacterial
cancer Clavibacter michiganensis subsp. michiganensis, bacterial
black spotting Xanthomonas vesicatoria and bacterial speck of tomato plants Pseudomonas syringae pv. tomato. It was revealed that
the most active against the agents of bacterial black spotting and
bacterial cancer are biologicals Phytocide and Phytohelp, based on
the bacteria Bacillus subtilis, that are recommended by us for use in
the agriculture.
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