Chitosan Inhibits the Growth of Phytophthora botryosa: The Causal Agent of Para Rubber Leaf Fall Disease
The most destructive disease of Para rubber seedling is a leaf fall caused by Phytophthora botryosa. The bioactivity of chitosan against P. botryosa infection was studied in vitro. The growth rates of P. botryosa in potato dextrose agar were determined with different concentrations of chitosan. A chitosan concentration of 0.5 mg mL-1 was sufficient to cause clear growth inhibition (about 87% reduction in diameter). Mycelia and oogonia wilt were observed in scanning electron micrographs of P. botryosa. The biodegradable chitosan therefore inhibits the in vitro growth of P. botryosa and may have potential for practical disease management of leaf fall.
Received: February 01, 2013;
Accepted: March 06, 2013;
Published: June 24, 2013
Para rubber is one of the most important economic crops in Thailand. The major
rubber-production areas are in southwest and northeast of Thailand. Like most
other cultivated crops, rubber is facing serious problems from several diseases:
after long periods of high rainfall, the leaf fall disease becomes a problem.
Two species of Phytophthora are known as destructive diseases in rubber:
both Phytophthora palmivora and P. botryosa are common.
The infection process of a soil borne pathogen, Phytophthora, starts
at an immature pod resulting in pod rot and becomes the source of inoculum (Chantarapratin
et al., 2001). Spores of the pathogen are spread by rain splash from
the infected leaves to the trapping panel (Johnston, 1989).
In Thailand, leaf fall epidemics occur from June to December (Chantarapratin
et al., 2001). An obvious sign of Phytophthora caused leaf
fall is the coagulated latex in a central lesion of the petiole (Fig.
1). However, lesions can occur anywhere along the length of the petioles.
Heavy defoliation may lead to dieback of terminal branches (Chee,
1968; Turner, 1969; Johnston,
Application of fungicides, such as metalaxyl and fosetyl-Al, is the most effective
method to control Phytophthora disease. However, chemical control may
select for fungicide-resistant strains of these pathogens and is under review
in many countries also due to human health concerns. Bioactive substances are
actively pursued for an alternative approach.
||Para rubber leaf fall symptoms. The white arrows indicate
coagulated latex in central lesions on petioles
The application of chitosan or derivatives of chitosan has been effective as
a biological control against several fungi in several hosts (Allan
and Hadwiger, 1979; Hirano and Nagao, 1989; Kendra
et al., 1989; Stossel and Leuba, 1984).
Chitosan has reduced the disease severity and incidence of Puccinia pimpinellae
(Saber et al., 2009) and it had inhibitory effects
on soil borne phytopathogenic fungi (Stossel and Leuba,
1984; Hernandez-Lauzardo et al., 2011).
For these reasons the native form of low molecular weight chitosan was selected
to test its activity against the causal agents of para rubber leaf fall disease.
Agar plates were prepared with various concentrations of chitosan and the mycelia
and oogonia grown on the plates were also characterized from Scanning Electron
Microscope (SEM) micrographs.
The objective of this study was to test the antimicrobial effects of chitosan
on P. botryosa, the causal agent Para rubber leaf fall disease, in
MATERIALS AND METHODS
Cultures and growth conditions: The P. botryosa was kindly provided
by Asst. Prof. S. Chuenchit (Department of Pest Management, Faculty of Natural
Resources, Prince of Songkla University). The fungi were stored on Potato Dextrose
Agar (PDA) (HiMedia, Mumbai, India) slants at 4°C. The fungal culture was
routinely maintained on PDA made of 200 g potato, 20 g dextrose and 15 g agar,
at room temperature (25-30°C) for 5 days, prior to testing with various
concentrations of chitosan solution.
Preparation of chitosan: Low molecular weight (10253 kDa) chitosan was
purchased from Aldrich, China. Purified chitosan was prepared as described by
Benhamou et al. (1998). The stock solution (1%,
w/v) of chitosan was prepared by dissolving purified chitosan in 0.5% (v/v)
glacial acetic acid under continuous stirring and the pH was adjusted to 5.6
using 1 N NaOH (Du et al., 1998). The chitosan
solution was autoclaved (120°C, 20 min) prior to use in assays.
Effect of chitosan on radial growth: To evaluate the effect of chitosan
on Phytophthora growth, PDA plates were amended with chitosan at different
concentrations (0.125, 0.25, 0.5, 1 and 2 mg mL-1) as by Laflamme
et al. (2000). Unamended PDA plates with 0.05% final concentration
of acetic acid (pH 5.6) served as negative controls. Comparison to these controls
shows the activity of chitosan and excludes the effects of acidic conditions.
The most prevalent para rubber pathogenic microorganism, P. botryosa,
was assessed for its growth inhibition. Five replicate plates at each chitosan
concentration were inoculated in the center with a plug (5 mm diameter) from
the edge of a 3-5 day-old-colony of P. botryosa. The colony radii were
measured 7 days after the inoculations. The experiments were repeated twice.
Percent Inhibition of Diameter Growth (PIDG) was calculated using the following
where, A is diameter of control colonies, B is diameter of treated colonies.
Statistical analyses: A Complete Randomized Design (CRD) was repeated
twice for each experiment. Statistical analyses were run with SPSS software.
Prior to such analyses, the growth effects of chitosan treatments were normalized
to percentages relative to control. The differences between means from chitosan
treatments and controls were tested for statistical significance by Duncans
Multiple Range Test (DMRT) for multiple comparisons.
Microscopic analysis: Mycelia and oogonia of P. botryosa, either
untreated or treated with chitosan, were fixed by immersion in 2.5% glutaraldehyde
in 1 mol L-1 Phosphate Buffer (PB) at 4°C. They were then fixed
with 0.1% osmium tetroxide in PB for 30 min at room temperature. The samples
were dehydrated gradually with alcohol solutions (50, 70, 80, 95 and 100%) and
then air-dried for one week before coating with gold/palladium. Finally they
were transferred to Scanning Electron Microscope (SEM) stubs. Three random fields
of view per sample were photographed.
RESULTS AND DISCUSSION
Chitosan solutions in five concentrations (0.125, 0.25, 0.5, 1 and 2 mg mL-1)
were tested for their inhibitory effect of P. botryosa linear growth.
The antimicrobial activity of chitosan against P. botryosa is shown in
Fig. 2 and Table 1. All concentrations reduced
the growth of P. botryosa at 7 days post inoculation and the growth inhibition
consistently increased with concentration. Effective inhibition was obtained
with 0.5, 1 and 2 mg mL-1 (87.5, 90.6 and 91.3% linear growth reduction,
respectively). When the concentration was 0.5 mg mL-1, the antimicrobial
activity of chitosan was already at an acceptable level. The PDA amended with
0.05% acetic acid served as control. No growth differences were observed between
this control and plain medium without acetic acid (data not shown).
Statistical analysis of the antifungal effect of chitosan concentration against
P. botryosa is shown in Table 1. The activity increases
with chitosan concentration, with the highest concentrations having a significantly
different (p<0.01) effect from the lowest ones (and control).
||Effects of different concentrations of chitosan on radial
growth of P. botryosa
|% inhibition rate is relative to corresponding control. Different
superscripts indicate values that are significantly different (p<0.01)
|| Effects of chitosan on radial growth of P. botryosa.
PDA amended with 0.05% acetic acid served as (a) A control, for comparison
with chitosan solutions at (b) 0.125, (c) 0.25 and (d) 0.5 1 mg mL-1
The plant pathogenic fungus-like organism P. botryosa was clearly sensitive
to chitosan, so these in vitro results suggest that chitosan may be an
effective growth inhibitor of P. botryosa.
Chitosan has been reported as an effective biocompound against several bacterial
and fungal strains (Liman et al., 2011). The
results of the present study demonstrate that the plant pathogenic fungus-like
organism P. botryosa is also sensitive to chitosan. Chitosan has great
potential as a biodegradable substance. Recent studies have shown that chitosan
is not only effective in inhibiting the growth of the pathogen but also in eliciting
activities (Benhamou, 1996; El Ghaouth
et al., 1999; Barka et al., 2004).
It has been shown that mycelia growth of fungi is inhibited by chitosan. The
level of inhibition was highly correlated with chitosan concentration (in the
range 0.75-6.0 mg mL-1), decreasing the radial growth of Alternaria
alternata, Botrytis cinerea, Colletrotrichum gloeosporioides
and Rhizopus stolonifer (El Ghaouth et al.,
1992b). The same effect was found on Rihzoctonia solani (Elmer
and LaMondia, 1994) and Sclerotium sclerotiorum (Cheah
et al., 1997). Furthermore, seed coating with modified chitosan
has inhibited Sphacelotheca reiliana, a causal agent of head smut of
corn (Zeng et al., 2010). The results on the
radial growth of P. botryosa (a fungus-like organism) with full growth
inhibition in 0.5% chitosan-amended PDA, are in agreement with to these prior
Examination of mycelia and oogonia from P. botryosa exposed to chitosan
(0.5 mg mL-1) showed that they had suffered. SEM images of 0.5% chitosan
treated and untreated mycelia and oogonia are shown in Fig. 3.
Antimicrobial effects of chitosan are seen on P. botryosa as reduction
of growth and wilt (Fig. 3b, d, f),
when compared to control (Fig. 3a, c, e).
||Scanning electron micrographs of Phytophthora botryosa
in PDA (a, c and f) and in PDA amended with 0.5 mg mL-1 of chitosan
(b, d and f)
Several fungi treated with chitosan had changes of morphology of the hyphae,
when observed by microscopy. In some experiments Fusarium oxysporum
f. sp. redicis-lycopersici, R. stolonifer and S. sclerotiorum
treated with chitosan showed abnormal hyphae shape and size reduction (Benhamou
and Theriault, 1992; El Ghaouth et al., 1992a,
b; Cheah et al., 1997).
Large vesicles or empty cells devoid of cytoplasm in the mycelium of B. cinerea
and F. oxysporum f. sp. albedinis were also observed (Barka
et al., 2004; El-Hassni et al., 2004).
In this study, SEM micrographs showed reduction of size of P. botryosa
as well as wither of mycelia and oogonia. Further studies are planned to follow
up on this in vitro study, by assessing the management of leaf fall disease
by chitosan application, in a greenhouse or in the field.
As a summary of current results chitosan, as a bioactive compound, directly
inhibits the in vitro growth of P. botryosa and withers its mycelia
This study was supported by the grants from the Prince of Songkla University,
grant number NAT550131S. The copy-editing service of Research and Development
Office (RDO) of Prince of Songkla University (PSU), especially the comments
and suggestions of Assoc. Prof. Seppo Karrila, are gratefully acknowledged.
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