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 formula:

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 Duncan’s 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).

Table 1:	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)

Fig. 2(a-d):	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 observations.

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).

Fig. 3(a-f):	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 and oospores.

ACKNOWLEDGMENT

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.