Use of Chitin and Chitosan Against Tomato Root Rot Disease under Greenhouse Conditions

Research Journal of Agriculture and Biological Sciences, 2(4): 147-152, 2006
© 2006, INSInet Publication
Use of Chitin and Chitosan Against Tomato Root Rot
Disease under Greenhouse Conditions
Abd-El-Kareem, F., Nehal S. El-Mougy, Nadia G. El-Gamal and Y.O. Fotouh
Department of Plant Pathology, National Research Centre, Giza, Egypt
Abstract: Root rotcaused byRhizoctonia solani, Fusarium solani and Sclerotiumrolfsii is themost destructive
disease of tomato plants. Effect of chitin and chitosan on root rot pathogens as well as their influence on soil
microflora and tomato root rot disease incidence under laboratory and greenhouse conditions were studied.
Chitosan at 6 g/l completely inhibited the linear growth of all tomato root rot fungi tested, while chitin has no
inhibitory effect. Tomato root rot fungi were highly affected by the presence of chitin or chitosan in the soil.
Their counts were decreased throughout the experiment period comparing with untreated soil. Effects of chitin
on fungal counts increased gradually by prolonging the experiment period from 15 up to 45 days, while the
opposite feature was observed with chitosan treatment. As for chytinolytic bacteria and actinomycetes, all
concentrations of both chitin or chitosan caused high increase in total count of chitinolytic bacteria. Bacterial
counts were greater in all concentrations of chitin and chitin plus chitosan than chitosan only. The bacterial
counts were increased as the period of experiment increased to reach its maximum after 45 days of treatments.
It was noticed that Bacillus spp. was the most dominant genus of isolated bacteria. Meanwhile, actinomyces
was not affected with all treatments for all periods. All treatments increased the chitinase activity in tomato
plants. The most effective treatment was chitin plus chitosan (6 g/kg soil), which increased the activity of
enzyme more than 148% as compared with untreated plants followed by chitin or chitosan individually at
6 g/kg soil, which increased the chitinase activity more than 100%. Under greenhouse greenhouse conditions,
allconcentrations of chitin or chitosanwere significantly reduced the tomato root rot incidence comparing with
untreated plants. The highest reduction was obtained with chitosan or chitin at 6 g/kg soil as single treatments
which reduced the disease incidence more than 88.0, 86.2 and 83.1% in infested soil with R. solani, F. solani
and S. rolfsii, respectively. Combination between chitin followed by chitosan (6 g/kg soil of both) reduced the
disease incidence by 94.4, 92.9 and 87.2% in infested soil with R. solani, F. solani and S. rolfsii, respectively.
Increasingconcentrations of either chitin or chitosan in combination cause more reduction in disease incidence.
It could be suggested that combined treatments between chitin and chitosan might be used commercially for
controlling tomato root rot diseases under field conditions.
Key words: Chitin, Chitinase, Chitosan, Microflora, Root rot, tomato
INTRODUCTION
resistance against soilborne fungi[11-14]. Field application
of Chitosan for inducing resistance against late and early
Tomato plants is theone of most important vegetable
blight diseases of potato and root rot disease of lupin
crops in Egypt and other countries. Root rot disease
plants was also reported by Abd-El-Kareem et.al.,[15-17].
caused by Rhizoctonia solani Kuhu.; Fusarium solani
Several studies have demonstrated that over expression of
(Mart) Sacc. and Sclerotium rolfsii Sacc. is the most
chitinasesand B-1,3-glucanase in plants is associatedwith
destructive disease of tomato[1,2]. Controlling such
enhanced resistance to various fungal pathogens[18-21].
diseases mainly depend on fungicides treatments[3].
The purpose of the present work was designed to
However,fungicidal applications cause hazards to human
evaluate the effect of chitin and chitosan singly or in
health and increase environmental pollution. Therefore,
combined treatments on the growth of tomato root rot
alternative treatments for control of plant diseases are
pathogens as well as their effect on soil microflora and
needed[4]. Chitin was reported as resistance inducer
tomato root rot disease incidence under greenhouse
against soilborne diseases[5-7].
conditions.
Addition of small quantities of chitin to soil resulted
in a marked reduction in root rot diseases of some
MATERIALS AND METHODS
plants[6-8]. Furthermore, chitosan is a safe material which
has antifungal activity against many plant pathogens[9,10]
Source of pathogenic fungi and tomato seeds: Tomato
Chitosan is a non-toxic compound was reported to induce
pathogenic fungi, i.e. R. solani; F. solani and S. rolfsii as
Corresonding Author: F. Abd-El-Kareem, Department of Plant Pathology, National Research Centre, Giza, Egypt.
E-mail: Ford62nrc@yahoo.com
147

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Res. J. Agric. & Biol. Sci., 2(4): 147-152, 2006
the causal agents of tomato root rot diseases were kindly
Effect of chitin andchitosan on tomato root rot disease
obtained from Plant Pathology Dept., National Research
under greenhouse conditions
Centre, Giza, Egypt. Meanwhile, tomato seeds cv. Kastel
Soil infestation with pathogenic fungi: One pathogenic
rock were obtained from Vegetable Crops Research
isolate of each R. solani; F. solani and S. rolfsii was
Department, Agricultural Research Centre, Giza, Egypt.
grownon sandy–barley medium (1:1 w:wand 40% water)
for 20 days at 25±2oC. Natural sandy loam field soils were
Effect of chitin and chitosan on the growth of tomato
artificially infested individually with prepared fungal
root rot fungi: The inhibitory effect of chitin and
inoculum at the rate 3% of soil weight, then mixed
chitosan (Sigma company) against tomato root rot fungi
thoroughly.
was tested in Vitro at four concentrations, i.e. 0, 2, 4 and
6 g/l. Chitin or chitosan were added to conical flasks
Soil amendments with chitin and chitosan: Chitin or
containing sterilized PDA medium before its solidifying
chitosan at four concentrations, i.e. 0, 2, 4 and 6 g/kg soil
and rotated gently then disbanded into sterilized Petri-
were tested against tomato root rot incidence. Previously
plates (9 cm diameter). Plates were individually
infested soil was mixed individually with each
inoculated at the centre with equal disks (6 mm diameter)
concentration tested of chitin or chitosan.
taken from 10 days old cultures o f each F. solani;
R. solani and S. rolfsii, then incubated at 25±2°C. Linear
Soil amendments with combined treatments between
growth of tested fungi was measured when the
chitin and chitosan: Infested soils with tomato root rot
control plates (medium free of Chitinor chitosan) reached
pathogens were mixed individually with chitin at
full growth and the average growth diameter was
concentrations of 0, 3 or 6 g/kg soil. Ten days later
calculated. Each treatment was represented by 5 plates as
chitosan was added in alternative order to the same soil at
replicates.
the same concentrations.
Treated infested soils were poured into plastic pots
Effect of chitin and chitosan as soil amendment on
(30 cm diameter) and irrigated every other day for ten
total count of pathogenic fungi, actionmyces and
days. Tomato transplants cv. Kastel rock were then
chitinolytic bacteria: The influence of chitin and
planted at the rate of 6 transplants/pot and 6
chitosan on some soil microflora was evaluated at four
pots/treatment were used. The percentage of tomato root
concentrations, i.e. 0, 2, 4 and 6 g/kg soil in addition to
rot incidence was recorded up to 50 days of transplanting
combined treatments between chitin and chitosan at
date.
6 g/kg (of each) were mixed individually with previously
infested soil with R. solani, F. solani or S. rolfsii. Plastic
Statistical analysis: Tukey test for multiple comparisons
pots (30 cm diameter) were filled with treated infested
among means was utilized[26].
soils, then tomato transplants cv. Kastel rock were
planted. The rhizospheric microflora as a total count of
RESULTS AND DISCUSSIONS
pathogenic fungi, actinomycetesand chytinolytic bacteria
after 15, 30 and 45 days of transplant date were estimated
Effect of chitin and chitosan on the growth of tomato
according to methods of Lauw and Webly[22] and
root rot fungi: The inhibitory effect of chitin and
Kobayashi et.al.,[23].
chitosan on the growth of tomato root rot fungi, i.e.
F. solani; R. solani and S. rolfsii was tested. Results in
Effect of chitin and/or chitosan as soil amendments on
Table 1 indicate thatchitin has no inhibitory effect on the
chitinase activity of tomato plants: The same
growth of all tested fungi. On the other hand, all
abovementioned treatments of chitin or chitosan were
concentrationsof chitosan hadsignificantinhibitory effect
evaluated for their effect on chitinase activity of tomato
against tested fungi. Chitosan at 6 g/l completely
plants as follows:
inhibit the linear growth of all tomato root rot fungi. The
Extraction of enzyme: Chitinase activity was determined
Table 1: Linear growth of tomato root rot fungi as affected by
after 10 days of seedlings planted. The enzyme extracted
different concentrations of chitin or chitosan
Average linear growth (mm)
from tomato plants and the supernatant was prepared
Concentration
----------------------------------------------
according to method of Tuzun et.al.,[24]. The chitinase
Treatment
(g/ kg soil)
R. solani
F. solani
S. rolfsii
activity was determined by colourimetric method of
Chitin
2
90.0a
90.0a
90.0a
Boller and Mauch[25]. Colloidal chitin was used as
4
90.0a
90.0a
90.0a
6
90.0a
90.0a
90.0a
substrate and dinitrosalicylic acid as reagent to measure
Chitosan
2
44.0b
50.0b
62.0b
reducing sugars. Chitinase activity was expressed as mM
4
23.0c
30.0c
36.0c
N-acetylglucose amine equivalent released/gram fresh
6
0.0d
0.0d
0.0d
Untreated
0
90.0a
90.0a
90.0a
weight tissue/60 minutes.
Figures with the same letter are not significantly different (P= 0.05)
148

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Res. J. Agric. & Biol. Sci., 2(4): 147-152, 2006
Table 2: Effect of single or combined treatment of chitin and chitosan on reduction (%) in total counts of pathogenic fungi in soil.
Reduction in total count of pathogenic fungi %
---------------------------------------------------------------------------------------------------------------------------
R . solani
F. solani
S. rolfsii
---------------------------------------------------------------------------------------------------------------------------
Days after treatment
Concentration
--------------------------------------------------------------------------------------------------------------------------------
Treatment
(g/kg soil)
15
30
45
15
30
45
15
30
45
Chitin
2
10.5
20.4
30.5
8.4
17.5
32
10.2
12.4
25.2
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Chitin
4
23.4
45.6
65
20.3
43.2
60
8.5
40
50
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Chitin
6
24.7
50.4
63
19.5
40.2
63
12.0
33
55
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Chitosan
2
50
45
40.5
40
35.5
32
28.0
28
24
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Chitosan
4
70
65
60
65.2
58
54
55.5
50.2
52
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Chitosan
6
75.5
75.5
65
80
78
68
64.2
60.5
63
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Chitin+chitosan
6 +6
75
80
80
70.5
70.5
80
65.0
64
70
Table 3: Increase (%) in total count of chytinolytic bacteria and actinomyses in the rhizosphere of tomato plants in response to single or combined
treatment of chitin and chitosan.
Increase %
----------------------------------------------------------------------------------------------------------------------------
Actinomycetes
Chitinolytic bacteria
-----------------------------------------------------------------------------------------------------------------------------
Days after treatment
Concentration
----------------------------------------------------------------------------------------------------------------------------
Treatment
(g/ kg soil)
15
30
45
15
30
45
Chitin
2
0
0.0
0.0
10.5
30
43
-----------------------------------------------------------------------------------------------------------------------------------------------------
4
0.0
2.0
2.0
15.4
52
62
-----------------------------------------------------------------------------------------------------------------------------------------------------
6
0.0
0.0
2.0
20
55
66
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Chitosan
2
0.0
0.0
2.0
0
10
12
-----------------------------------------------------------------------------------------------------------------------------------------------------
4
0.0
0.0
0.0
10
20
20
-----------------------------------------------------------------------------------------------------------------------------------------------------
6
0.0
0.0
0.0
8
22
19
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Citin + chitosan
6+6
0.0
2.0
2.0
12
53
60
moderate effect was obtained with chitosan at 4 g/l which
fungi. While, chitosan caused higher reduction in total
reduced the linear growth more than 60.0% for all tested
count of fungi after 15 days of treatments and then
fungi as compared with untreated fungal growth medium.
decreased as the period increased.
Meanwhile, chitosan at 2 g/l was less effective in this
concern.
Effect on chitinolytic bacteria and actinomycetes:
Results in Table 3 indicate that, all concentrations of both
Effect of chitin and chitosan as soil amendment on
chitin or chitosan caused high increase in total count of
total count of pathogenic fungi, actionmyces and
chitinolytic bacteria. Bacterial counts were greater in all
chitinolytic bacteria
concentrations of chitin and (chitin+ chitosan) than
Effect on tomato root rot pathogenic fungi: Results in
chitosan only. The bacterial count was increased as the
Table 2 indicate that tomato root rot fungi were highly
period ofexperiment increased to reach its maximumafter
affected by the presence of chitin or chitosan in soil,
45 days of treatments. It noticed that Bacillus spp. was the
whereas their counts decreased throughout
the
most dominant genus of isolated bacteria. Meanwhile,
experimental periods comparing with untreated soil. It
actinomyces was not affected with all treatments at all
was noticed that the influence of chitin treatment on
experimental periods.
fungal counts increased gradually by prolonging the
experimental period from 15 up to 45 days, while the
Effect of chitin and/or chitosan as soil amendments on
opposite trend was observed with chitosan treatment.
chitinase activity of tomato plants: Results in Table 4
Chitin treatments caused slight reduction after 15 days
indicate
that all treatments increased the chitinase
and increased as the experimental period increased for all
activity. The most effective treatment was chitin plus
149

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Res. J. Agric. & Biol. Sci., 2(4): 147-152, 2006
Table 4: Chitinase activity of tomato plants as affected by grown in soil treated with different concentrationsof chitin and chitosan under greenhouse
conditions
Soil infestation
--------------------------------------------------------------------------------------------------------------------------
R. solani
F.solani
S. rolfsii
------------------------------
---------------------------------
--------------------------------
Concentration
Chitinase
Increase
Chitinase
Increase
Chitinase
Increase
Treatment
(g/kg soil)
activity
(%)
activity
(%)
activity
(%)
Chitin
2
4
60
5
100
4.5
80
-----------------------------------------------------------------------------------------------------------------------------------------------
4
4.2
68
5.2
108
5.0
100
-----------------------------------------------------------------------------------------------------------------------------------------------
6
5.4
116
5.5
120
5.0
100
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Chitosan
2
4
60
4.5
80
4.2
68
-----------------------------------------------------------------------------------------------------------------------------------------------
4
4.5
80
5
100
4.3
72
------------------------------------------------------------------------------------------------------------------------------------------------
6
5
100
5.5
120
5.5
120
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Chitin+chitisan
6+6
6.2
148
6.5
160
6.3
152
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
untreated
0
2.5
-
2.5
-
2.5
-
Chitinase activity was expressed as mM N-acetylglucose amine equivalent released/gram fresh weight tissue/60 minutes
shitosan (6 g/kg soil), which increased the activity of
Table 5: Root rot incidence in tomato plants as affected by different
enzyme between 148 and 160% as compared with
concentrations of chitin or chitosanas soil amendments under
untreated plants, followed by chitin or chitosan
greenhouse conditions.
Tomato root rot incidence %
individually at 6 g/kg soil, which increased the chitinase
Concentration
---------------------------------------------
activity more than 100%, while other treatments have
Treatment
(g/ kg soil )
R. solani
F. solani
S. rolfsii
moderate effect.
Chitin
2
24.5b
23.4b
35.5b
4
12.0c
15.0 c
19.2c
6
8.0c
8.8d
13.5d
Effect of chitin and chitosan on tomato root rot disease
Chitosan
2
22.0b
26.0b
35.5b
under greenhouse conditions: Results in Table 5
4
11.5c
16. 5c
17.0c
indicate that all concentrations of chitin or chitosan
6
8.4c
9.0d
11.5d
significantly reduced the tomato root rot incidence
Untreated
0.0
70.0 a
65.0a
80.1a
Figures with the same letter are not significantly different ( P= 0.05)
comparing with untreated soil. Data also, show that the
percentage of root rot incidence decreased significantlyby
Table 6: Effect of combined treatment between chitin and chitosan as
increasing concentrations of either chitin or chitosan. The
soil amendments on tomato root rot diseases under
highest reduction wasobtained with chitosan or chitin was
greenhouse conditions
Treatment
at 6 g/kg soil treatment which reduced the disease
-----------------------------
Root rot diseases %
incidence more than 88.0, 86.2 and 83.1% in infested soil
Chitin
Chitosan
----------------------------------------------
with R. solani, F. solani and S. rolfsii, respectively
(g/kg soil)
(g/kg soil)
R. solani
F. solani
S. rolfsii
followed by concentration at 4 g/kg of both which
Chitin 3
0
25.0b
23.0b
32.0b
3
16.2c
13.0c
22.0c
reduced the disease incidence more than 74.6% as
6
10.0d
9.0cd
14.0d
compared with untreated plants. Meanwhile chitin or
Chitin 6
0
12.5cd
14.0c
21c
chitosan at 2 g/kg showed moderate effect.
3
10.5d
9.0cd
17.0d
6
4.5e
5.0d
9.0e
0.0
0
80.0a
70.5a
70.4a
Effects of combined treatments between chitin and
3
24.4b
20.0b
30.0b
6
10.2d
12.0c
18.0cd
chitosan as soil amendments for controlling tomato
Figures with the same letter are not significantly different ( P= 0.05)
root rot under greenhouse conditions: Results in
Table 6 indicate that significant reduction was observed
disease incidence. Combined treatments between chitin at
for tomato root rot incidence when soil was treated with
3 or 6 and chitosan at 6 or 3 g/kg soil, respectively
both or each of chitin and chitosan. Combination
reduced the root rotdisease more than 75.8% for alltested
treatment as chitin followed by chitosan (6 g/kg soil for
fungi. Meanwhile single treatment showed moderate
each) reduced the disease incidence by 94.4, 92.9 and
effect.
87.2% in infested soil with R. solani, F. solani and
Tomato plants is the one of most important vegetable
S. rolfsii,respectively. Increasing concentrations of either
crops overall the world which seriously attacked by
chitin or chitosan in combination cause more reduction in
soilborne fungi, i.e. Rhizoctonia solani, Fusarium solani
150

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Res. J. Agric. & Biol. Sci., 2(4): 147-152, 2006
and Sclerotium rolfsii inducing root rot diseases. Some
soil with chitin amendments was also r eported by
natural and safe alternatives have been reported for
Bell et.al.,[7], Godoy et.al.,[32] and Mian et al.,[33]. On the
replacing usages fungicides for controlling such diseases.
other hand, the recorded reduction in root rot incidence in
In this regards, Chitin and chitosan the safe materials
present study might be due to chitin treatments elicitor
which werereported to induceresistanceagainst soilborne
defense response in plants[6]. Also, the recorded results
diseases[6-8,14]. In the present study, under in vitro
indicate that chitin treatments caused high increase in
conditions chitin at concentrations of 2, 4 and 6 g/l
chitinase activity. In this respect Kuchitsu et.al.,[6]
showed no inhibitory effect against tested fungi, while
reported that chitin fragments appear to elicit host
chitosan at 6 g/l completely inhibit the linear growth of all
responses through rabid and transient membrane
tested fungi. The inhibitory effect of chitosan against
depolarization. Moreover, chitin was reported to be used
pathogenic fungi was reported by Hirano et.al.,[9] and
as soil fertilizer[5,34].
Abd-El-Kareem[14]. In this respect, two models have been
On the light of the present study, it could be
proposed to explain the anti-fungalactivity ofchitosan i.e.
suggested that the use of natural safe materials, i.e. chitin
the activity of chitosan is related to its ability to interfere
and chitosan as fungicides alternatives is considered one
with the plasma membranefunction[27] and the interaction
of low cost and effective applicable methods for
of chitosan with fungal DNA and RNA[28]. Addition of
controlling suchsoilborne plantpathogenscausing tomato
small quantities of chitin to soil resulted in a marked
root rot diseases.
reduction in root rot diseases of some plants[6,8]. Also,
chitosan was reported to induce resistance against
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