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FTIR spectroscopic analysis of luminescent mushroom (Neonothopanus nambi Speg.) induced disease resistance against tomato root-knot nematode, Meloidogyne incognita Chitwood.

Root-knot nematode, Meloidogyne incognita is widespread and important pest of economic crops around the world, hence it causes severe damage to field, horticultural and vegetable crops particularly to tomato production (1). This nematode can attack tomato in many part of the world (2) and caused yield loss of tomato estimated 28-47% annually (3). In order to control or manage the root-knot nematode, chemical control was effective, however it may cause toxic contamination to environment. Therefore, biological control of nematodes is considered as a potential management and effective alternative of nematicides (4).

Nematophagous fungi are soil-living fungi potentially as biocontrol agents of various plant parasitic nematodes (5). Many fungi can produce nematicidal compounds (6). In particularly, the fungus Omphalotus olearius, a luminescent mushroom that produced omphalotin A, showing nematicidal property against the root-knot nematode, M. incognita (7). Engler et al. (8) reported that the secondary metabolite isolated from culture filtrate of O. olearius affecting the growth and development of M. incognita. In Thailand, there were studies on the effectiveness of luminescent mushroom royal given name in Thai by Princes Maha Chakri Sirindhorn as "Hed Sirin Ratsamee", Neonothopanus nambi (9). The application of culture filtrate, spawn and bioactive compound of N. nambi were reported preliminary as having nematicidal effect and suppressed the root-knot disease incidence in tomato plants (9). Bioactive compounds of this mushroom was extracted and reported on their properties in various evaluations (10,11). However, previous application of these fungi was reported only beneficial to disease management involved toxic compound production or antagonism directly to the pathogen. It is interesting, therefore, that the luminescent mushroom, N. nambi may play a role in inducing systemic resistance in tomato plants against root-knot nematode.

Infrared spectroscopy or Fourier transform infrared (FTIR) is a simple and rapid technique that recently used for monitoring of chemical modification of cell wall components (12). FTIR spectroscopy provides more detailed chemical structures and composition of plant tissues including roots, stems and leaves, based on specific vibration characteristics of chemical functional group of each sample (13). Recently, the use of FTIR spectroscopy to identify the chemical structure of plant secondary metabolites has increasingly important within plant pathology research (14). However, the study on induced resistance by using N. nambi against root-knot nematode with FTIR has not been reported. Therefore, the objective of this study is to apply the FTIR spectroscopic analysis for assisted evaluation on luminescent mushroom, N. nambi against M. incognita in tomato.


Source of the luminescent mushroom (Neonothopanus nambi)

Luminescent mushroom (N. nambi) PW2 isolated from the Plant Genetic Conservation Project under Royal Initiation by Her Royal Highness Princess Maha Chakri Sirindhorn at Kok Phutaka, Wiang Kao District, Khon Kaen Province, Saksirirat et al. (15) was used for all experiment. The mushroom hyphae were maintained on potato dextrose agar (PDA) at 28[degrees]C. In this study, N. nambi fungus was prepared in three formulae, culture filtrate (CF), mycelia spawn (SP) and bioactive compound, aurisin A extracted according to (10). Culture filtrate of N. nambi were prepared on PDA at 28[degrees]C for 7 days before transfered of mycelium agar, 5 disks to 500 ml flasks containing 200 ml of Yeast Malt Broth (YMB). After 15 days of incubation at room temperature (25-30[degrees]C), tested fungus was filtered through Whatman No.1 filter paper to obtain culture filtrates. Spawn of N. nambi was prepared using sorghum grains boiled and autoclaved for 30 minutes. Hyphae of N. nambi were inoculated on steriled sorghum grains and incubated at 28[degrees]C for 20 days. Ten to twenty sorghum grains with fully colonized by N. nambi were inoculated on rubber tree sawdust supplemented with 10% of wheat flour and incubated at 28[degrees]C for 26 days before used. Bioactive compound of N. nambi were extracted from dry mycelia using method described by Buaart et al. (10). The powder of bioactive compound from N. nambi was dissolved in dimethyl-sulfoxid (DMSO) at 0.2% concentration with sterile distilled water. Bioactive compound was prepared of a concentrations 500 milligram per liter (mg/l) for further test.

Preparation of root-knot nematode (M. incognita) egg inoculums

Egg inoculum was prepared from tomato roots infected with M. incognita, which maintained in greenhouse of Department of Plant Science and Agricultural Resources, Faculty of Agriculture, Khon Kaen University, Thailand. The root-knot nematode eggs were extracted from roots with 0.5% NaOCl and collected using the modified technique of (16). The nematodes inoculums (3,000 eggs per pot) were used for each experiment.

Bioassay in greenhouse experiment

The effect of induced systemic resistant property of N. nambi was investigated by evaluation of root gall percentage and number of infective larvae (J2) (M incognita) using the split-root experimental design, which allows inoculation of the fungus and nematode at separated locations on the root system. Three-week-old tomato (Sida cultivar, PA Seed Company) seedlings were uprooted from sterilized soil, washed with tap water, and divided the roots into two halves with a sterilized dissecting scalpel. Each half of the root system was transplanted separately into plastic pots ([empty set] 12 cm) containing a sterilized mixture of clay soil and sand (1:1 v/v) 300 g soil/pot. One part of the root system was treated with different conditions of N. nambi (inducer roots). Soil treated with sterile water served as controls. Ten days after treatments, the other half of the root system was inoculated with 3,000 eggs of M. incognita (responder roots) by soil infestation. Complete randomized design (CRD) consisted of 7 treatments and 4 replicates were used (Table 1). Treatments were 1) non treated plants (control) either half-root system; 2) one half-root system added with 3,000 eggs of M. incognita (N); 3) nematode added into one half-root system and 0.2% DMSO added into another half-root system (0.2% DMSO); and 4) nematode added into one half-root system and treated with aurisin A (500 mg/L) added into another half-root system (aurisin A); 5) nematode added into one half-root system and the culture filtrate of N. nambi PW2 (30 ml) was added into another half-root system (CF PW2); 6) nematode added into one half-root system and the spawn of N. nambi PW2 (30 g) added into another half-root system (SP PW2) and 7) nematode added into one half-root system and the combination of culture filtrate (15 ml) and spawn of N. nambi isolate PW2 (15 g) were added into another half-root system (CF+SP PW2). NematodeM. incognita was applied at 3,000 eggs/half-root system. After 30 days, root galling percentage and numbers of J2 penetrating in roots were recorded. Root galling were evaluated according to; 0 = no root-knot, 1 = 1-25% of root-knot, 2 = 26-50% of root-knot, 3= 51-75% of root-knot, whereas 4= 76-100% of root-knot. Each half-root system (responder root) was stained in 0.01% acid fuchsin in lactophenol (17). Numbers of J2 infecting in tomato roots were quantified per 1 g. roots. Data were analysis of variance (ANOVA) and the differences between means were compared using Duncan's multiple range test (DMRT).

Biochemical analysis

Crude enzyme samples were prepared using 0.5 g of tomato root harvested at 10 days of interval after treated of N. nambi into soil without challenge inoculation with nematode. The root were homogenized with 1.2 ml of 0.1 M acetate buffer pH 5.0, centrifuge at 14,000 rpm then transferred supernatant to a new Eppendorf tube 1.5 ml and kept at -20[degrees]C until used. Protein content in enzyme solutions were determined by method of Bradford (18) using bovine serum albumin as a standard protein.

Assay of phenolic compound

Tomato root samples (0.5 g) were homogenized in 10 ml of 80% methanol and agitated for 15 min at 70[degrees]C (19). One ml of the methanolic extract was added to 5 ml of distilled water and 250 [micro]l of Folin-Ciocalteau reagent then the solution was incubated at 25[degrees]C. The absorbance of the developed blue color was measured using a spectrophotometer with wave length 725 nm. Catechol was used as the standard. The amount of phenolic compound was expressed as [mg.sup.-1] g fresh weight.

Assay of salicylic acid (SA)

Tomato root samples (0.5 g) were homogenized in 0.5 ml cold-ice buffer (90 ml methanol, 9 ml acetic acid and 1 ml of water) (20). Then 0.5 ml of 0.05 M ferric ammonium sulfate were added in reaction mixtures and incubated at 30[degrees]C 15 min. The absorbance of salicylic acid was measured with wave length 527 nm and compared with a standard curve of SA dissolved in ethyl acetate. The amount of SA was expressed as mg [g.sup.-1] FW.

Assay of polyphenol oxidase (PPO)

PPO activity was determined according to the method of (21). Tomato root samples (0.5 g) were ground in 2 ml 0.1 M sodium phosphate buffer (pH 6.5) and centrifuged at 16000 g for 15 min at 4[degrees]C. The supernatant was used as the enzyme source. The reaction mixture consisted of 0.2 ml of the enzyme solution and 1.5 ml of 0.1 M sodium phosphate buffer (pH 6.5). To start the reaction, 0.2 ml of 0.01 M catechol were added. The reaction mixture was measured the absorbance at 495 nm. The PPO activity was expressed as [micro]mol (quinone) [mg.sup.-1]protein/hr.

Assay of Phenylalanine ammonia-lyase (PAL)

PAL activity was determined according to the method of (22). The reaction mixture consisted of 0.2 ml of the enzyme solution and 0.5 ml of 12 mM L-phenylalanine in 0.1 M sodium borate buffer, pH 8.8 were incubated for 30 min at 30[degrees]C. PAL was determined as the rate of conversion of L-phenylalanine to trans-cinnamic acid. The reaction mixture was analysed by colorimetric method using spectrophotometer at 290 nm. PAL activity was expressed as [micro]mol (trans-cinnamic acid) [mg.sup.-1]protein/hr.

Assay of chitinase activity

Chitinase activity was evaluated by incubation of 1 ml 1% colloidal chitin and 1 ml enzyme solution in 0.1 M acetate buffer pH 5.0 at 37[degrees]C for 1 hour. Reaction mixture was stopped by boiling aliquot for 15 min. The amount of reducing sugar released in reaction mixture was calculated by analysis method of (23) using N-acetylglucosammine (GlcNac) as standard. Chitinase activity was expressed as [micro]mol (GlcNac) [mg.sup.-1]protein/hr.

Assay of protease activity

Protease activity was evaluated by incubation 0.1 ml enzyme solution and 1.0 ml 1% casein dissolved in 0.1 M phosphate buffer, pH 7.0 at 37[degrees]C for 1 hour. Reaction mixture was stopped by 3.0 ml of 10% trichloroacetic acid. The enzyme activity was measured the absorbance at 280 nm using tyrosine as standard according to the method of (24). Protease activity was expressed as [micro]mol (tyrosine) [mg.sup.-1]protein/hr.

FTIR analysis of tomato root

Tomato roots (10 mg) were oven-dried at 60[degrees]C for 3 days. The dried tomato roots were ground with a crystal mortar and pestle. FTIR spectra were acquired at the Synchrotron Light Research Institute (SLRI), Nakhon Ratchasima, Thailand, using a Bruker Vertex 70 FTIR spectrometer (Bruker Optics Inc., Ettlingen, Germany) equipped with a nitrogen cooled MCT (HgCdTe) detectors. Absorbance spectra were acquired in the spectral range of 4000-700 [cm.sup.-1]. FTIR samples were recorded in the reflection mode, 4 [cm.sup.-1] spectral resolution, 64 scans per spectrum using Pike MIRacle ATR germanium ATR (PIKE Technologies, Madison, USA).

Data processing and data analysis

Principle Component Analysis (PCA) was performed to distinguish different chemical components between treatment groups. The spectra were preprocessed by taking the second derivative using the Savitzky-Golay algorithm with nine points of smoothing and normalized with Extended Multiplicative Signal Correction (EMSC). PCA was performed using The Unscrambler software (version 9.7, CAMO Software AS, Oslo, Norway) employing the combined spectral ranges of3000-2800 [cm.sup.-1] and 1750-850 [cm.sup.-1]. Six PCs were selected for data analysis. Score plots (2D) were examined for any clustering of spectra and loading plots identified the spectral features explaining any observed clustering in the scores plots in each data set. The spectra of various treatment of tomato roots were subsequent clustered by Unsupervised Hierarchical Cluster Analysis (UHCA) using the Ward's algorithm method (OPUS 6.5 software; Bruker Optics Ltd., Ettlingen, Germany). UHCA was performed on a regions of 3000-2800 [cm.sup.-1] and 1750-850 [cm.sup.-1] with a total of 60 spectra.


Effect of N. nambi on inducing resistance root-knot disease of tomato

Split-root test revealed that luminescent mushroom, N. nambi, in all conditions could stimulate tomato resistant to root-knot nematode, M. incognita (Table 1) when treated the fungus 10 days before nematode egg infestation. The tomato treated with spawn (SP) 30g/pot or soil drenching by culture filtrate 15 ml/plant combined with spawn 15 g/pot (CF+SP) were the most effective for inducing resistance of tomato against root-knot nematode, they could reduce significantly (P<0.05) root gall in tomatoes at 3.5% (83.3% reduction) while the control root exhibited highest root galls (20.58%). Whereas, other treatment such as culture filtrate (CF) of N. nambi isolate PW2, 0.2% DMSO and aurisin A (500 mg/L) showed the lower percentage of root galls 4.5, 4.8 and 5.3%, respectively. These results indicated that N. nambi could reduce root galls 74-83%. On J2 penetration, treatment of culture filtrate combined with spawn of N. nambi isolate PW2 showed the best efficiency in reducing J2 penetration of 92.3% more than other treatments, culture filtrate, spawn, aurisin A and 0.2% DMSO reduced J2 penetration in root of tomato ranking 17.1-86.6%, while plants inoculated with only nematode (N-only) showed the maximum number of nematode juvenile in tomato roots of 1,036 J2/g root (Table 1).

Determination of defense enzymes by biochemical analysis Phenol assay

The results of induction of phenolic compound in tomato treated with N. nambi with different condition were determined in Table 2. The highest accumulation of tomato root (0.205 mg [g.sup.-1] fresh weight) were obtained from spawn (SP) followed by tomato root treated by aurisin A and culture filtrate (CF) with 0.128 and 0.093 mg [g.sup.-1] fresh weight, respectively compared to control treatment (Table 2).

Salicylic aicd assay

The result showed that SA level was significantly (P<0.05) higher in tomato roots treated with spawn (SP), culture filtrate combined with spawn (CF+SP) and culture filtrate (CF), with 0.268, 0.265 and 0.250 mg [g.sup.-1] fresh weight, respectively compared to control root (Table 2).

PAL assay

The highest activity of PAL enzyme was observed in tomato roots treated with culture filtrate combined with mycelia spawn (CF+SP) and mycelia spawn (SP) with 31.51 and 29.17 [micro]mol (trans-cinnamic acid) [mg.sup.-1] protein/hr compared with control root (Table 2).

PPO assay

All treatment of N. nambi increased level of PPO activity (Table 2). The highest activity of PPO was recorded in tomato roots treated with mycelia spawn (SP) with 14.15 [micro]mol (quinone) [mg.sup.-1] protein/hr followed by tomato root treated with mycelia spawn (SP), culture filtrate (CF), 0.2% DMSO, aurisin A, and culture filtrate combined with mycelia spawn (CF+SP) were 8.51, 8.15, 6.84 and 6.48 [micro]mol (quinone) [mg.sup.-1] protein/hr, respectively compared to control root.

Chitinase activity

The highest activity of chitinase enzyme were obtained in tomato treated with mycelia spawn (SP) with 5.23 [micro]mol (GlcNAc)/[mg.sup.-1] protein)/hr followed by tomato root treated with 0.2% DMSO were 3.93 [micro]mol (GlcNAc)/[mg.sup.-1] protein)/hr compared to control root (Table 2).

Protease activity

The highest activity of protease enzyme were measured in tomato treated with mycelia spawn (SP) with 26.05 [micro]mol (tyrosine)/[mg.sup.-1] protein)/ hr compared to control root (Table 2).

FTIR microspectroscopy

Tomato root samples obtained from six different treatments of N. nambi were examined by FTIR spectroscopy in order to monitor biochemical changes during induced resistance process. The average of all quality spectra after normalization and baseline correction over the range of 3000-2800 [cm.sup.-1] are showed in Fig 1(a). Significant spectral changes were observed in a peak position of 1400-1000 [cm.sup.-1] (Fig. 1a). The second derivative spectrum of such regions gives a negative peak, allowing easier identification of individual peaks in complex spectra (Fig. 1b). These spectral changes were associated with alterations in polysaccharide, cellulose and hemicelluloses. Tomato roots treated with N. nambi showed higher polysaccharide content region at spectral bands 1515 [cm.sup.-1] (aromatic skeleton of lignin, 1377 [cm.sup.-1] (C-H bending) which were assigned to cellulose, 1107 [cm.sup.-1] (C-C glycosidic link from pectin), and also clear spectral differences in carbohydrate-associated band 1060 [cm.sup.-1] and 1030 [cm.sup.-1] (C-O stretching) assigned to hemicelluloses when compared with untreated roots. The peak area integration of each absorption spectrum was further performed in order to presenting significantly differences between the untreated and treated roots (Table 3).

Our results indicated that the integral area of the tomato roots treated with mycelia spawn shows highest intensity of the cell wall protein region (1716-1560 [cm.sup.-1]) and also highest absorbance of lignin regions (1566-1495 [cm.sup.-1]) with 32.8 and 0.57%, respectively when compared with the untreated roots (28.2 and 0.40%), respectively. The integral area of tomato roots treated with aurisin A shows highest intensity of cellulose regions (1377 [cm.sup.-1]) with 33.2%, followed by culture filtrate (30.6%), and 0.2% DMSO (23.2%) than the untreated root (21.0%). The integral area of tomato root treated with 0.2% DMSO exhibited highest intensity of pectin regions (1178-1084 [cm.sup.-1]) with 31.5%, followed by culture filtrate combined with spawn (CF+SP) (25.1%) than the untreated roots (23.1%). The tomato roots treated with culture filtrate combined with spawn (CF+SP) also expressed highest intensity of hemicellulose regions (1087-975 [cm.sup.-1]) with 32.8 % than the untreated roots (27.3%) (Fig. 2). These different infrared absorbant spectra of cell wall protein and polysaccharide regions in the tomato roots treated with different condition of N. nambi might cause by differences in plant root responses related to induced resistance mechanisms.

To elucidate differences among these infrared spectra of tomato roots, absorption spectrum of each treatment was further analyzed with multivariate statistical techniques based on PCA to assess the spectral differences between average entire spectra as shown in Fig 3a. PCA was performed on second derivative spectra from all 160 spectra. The results showed clearly as separated with six clusters of tomato roots were classified with PC 1 versus PC2 score plot. The first two PCs explained 80% of the total variance in the data set (62% and 18% of the total variance explained by PC1 and PC2, respectively). Spectra from tomato roots treated with mycelia spawn (SP) and culture filtrate combined with mycelia spawn treated root (CF+SP) can be distinguished from other treatments by having positive PC1 scores (Fig. 3a). This spectra were highest negative value for PC1 loading (Fig. 3b) with variables between 1650 [cm.sup.-1], 1510 [cm.sup.-1], 1107 [cm.sup.-1], 1060 [cm.sup.-1] and 1030 [cm.sup.-1], possibly indicating that protein cell wall, lignin, and cell wall polysaccharide such as pectin and hemicelluloses are most responsible for discrimination. Moreover, spectra from other treatments such as tomato roots treated with culture filtrate (CF), 0.2% DMSO-treated root, aurisin A-treated root and untreated root are clearly separated by the negative correlation of PC1 (62%) (Fig. 3a), which have positive PC1 loadings (Fig. 3b) at 1378 [cm.sup.-1], suggesting a dominance of cellulose region. Whereas the spectra of mycelia spawn (SP)- and culture filtrate combined with mycelia spawn (CF+SP)-treated tomato root were clustered separately along PC2 (18%). The negative of PC2 loading of the glycosidic linkage (C-O-C) stretching at band 1030 [cm.sup.-1] attributed to hemicelluloses were used to separate the spectra of the tomato roots treated with spawn (SP) from tomato root treated with culture filtrate combined with spawn (CF+SP). In order to classify and determine the similarity of infrared spectra among the tomato roots both treated and untreated group, the Hierarchical cluster analysis (HCA) with Ward's algorithm were performed using spectral in the regions 3000-2800 [cm.sup.-1] and 1800-950 [cm.sup.-1] (Fig 4). There were two classes which could be distinguished between tomato roots treated with spawn (SP), culture filtrate combined with spawn (CF+SP) and root treated with culture filtrate (CF), 0.2% DMSO, aurisin A and untreated root below a linkage distance less than 6. Both principle component and cluster analysis indicated that the cell wall protein and polysaccharide regions were clearly discriminated among tomato roots treated with different conditions.


The split root experiment presented here was used to elucidate this possible involved mechanism of nematode suppression by luminescent mushroom, N. nambi. The results of the experiment revealed that soil treatment with spawn (SP) and culture filtrate combined with spawn (CF+SP) of N. nambi was found more effective in enhancing suppress of root-knot nematode, M. incognita in tomato. The application of spawn (SP) of N. nambi was most effective and significantly reduced root galling percentage and nematode penetration in tomato root when compared to other condition. These findings are in agreement with those of several workers (25,29). The work of (30) reported that the activity of nematophagous fungi may increase in response to sawdust to obtain nitrogen balancing in C/N ratio. However, mode of action of sawdust affected on nematophagous fungi is still not fully understood. Defense compounds and enzymes in host plants play an important role in the mechanism of resistance to nematodes. Involvement of biochemical compounds during induction of resistance in tomato was studied in enzyme extracts of tomato roots ten days after treatment of N. nambi. It could be concluded in this present study that tomato plants treated with the spawn of N. nambi enhances the induction of defense compounds and enzymes such as phenolic compound, salicylic acid, PAL, PPO, chitinase and protease than other treatments and untreated roots. These results are in agreement with previous studies of several researchers. Salicylic acid (SA) is a signal molecule involved in hypersensitive reactions in plants which important to activate defense reactions to root disease caused by nematode. Salicylic acid may activate plant enzymes to limit the nematode development and reproduction (31). The infestation of lesion nematode, Pratylenchus zeae was effected on PAL activity increasing in resistant cultivar of sugarcane when compared with susceptible cultivar (32). Brueske (33) also reported that the decreasing of PAL activity in susceptible tomato and carrot roots that infected by Meloidogyne spp. The increased activity of PAL may associate with the production of transcinnamic acid which acts as a substratum for lignin synthesis pathway towards protection against nematode penetration. Moreover, PAL is the most important enzyme playing a role for synthesis of phenols, phytoalexin and lignin. Enhanced activity of highest PPO activity has been reported in tomato that inoculated with M. incognita especially, in resistance cultivars (34). Other defense enzymes include pathogenesis-related proteins such as chitinase and protease were also reported the involvement of induced resistance against plant parasitic nematode. Chitinase may have some effect on the resistance mechanism of plants leading to a reduction of nematode by degrading of the cell wall and cause lysis of the cuticle cell of nematode. The highest accumulation of chitinase was observed in a study of (35) that the tomato cells treated with Pseudomonas flourescens strain Pf 128 which reduced nematode penetration in tomato root tissues. FTIR spectra analysis of tomato roots treated with different condition of N. nambi exhibited the absorption bands of cell wall proteins and cell wall polysaccharides in spectral range 1700-1000 [cm.sup.-1]. From the quantitative analysis of these components by integral area, it is found that higher cellulose content were observed from tomato root treated with bioactive compound extracted from N. nambi (aurisin A) than other treatments. Whereas, highest of cell wall protein and lignin accumulation were obtained from root treated with spwan (SP) of N. nambi. The results also confirmed by multivariate data analysis based on PCA and cluster analysis which exhibited that tomato root treated with spawn (SP) alone or root treated with culture filtrate combined with spawn (CF+SP) have more activation of cell wall components such as lignin, pectin and hemicelluloses than other treatments and untreated roots. Lignin is a cell wall component that covalently linked to hemicellulose and crosslinks different plant polysaccharides conferring mechanical strength to the cell wall (36), which plays on important role against many lytic enzymes produced by pathogens during host tissue colonization. In addition, the lignification in plants cell wall could be a crucial process in resistance to root-knot nematodes (37). The intensity of PAL, PPO, other defense enzymes and arise of cell wall protein and cell wall polysaccharides may be associated with the resistant mechanism present in the tomato against root-knot nematode. In conclusion, the present study implies that earlier and higher accumulation of phenols, salicylic acid and defense enzymes such as PAL, PPO, chitinase and protease and synthesized of cell wall polysaccharide in tomato roots treated with N. nambi involved in induced systemic resistance and resulting in significant reduction of nematode infection. Our study reported previously that the culture filtrate, spawn and the bioactive compound, aurisin A produced by N. nambi were effective biocontrol agents against root-knot nematode in tomato (10). This consequent result of the study reports for the first time on the induced resistance mechanism of luminescent mushroom, N. nambi to control the root-knot nematode,M. incognita in tomato. This indicated that tomato root applied with N. nambi in difference condition may exhibit different modes of induce resistance processes based on their infrared spectral changes. This response may leads to lignifications of cell walls of tomato root which is a mechanical barrier for the pathogens (38).


We would like to thank the Plant Genetic Conservation Project under Royal Initiation by Her Royal Highness Princess Maha Chakri Sirindhorn for supporting the research. Thanks are also extended to the the Center of Excellence on Agricultural Biotechnology, Science and Technology Postgraduate Education and Research Development Office, Commission on Higher Education, Ministry of Education (AG-BIO/ PERDO-CHE) and Agricultural Biotechnology Research Center for Sustainable Economy, including Cultivation and Product Development Research Group of Wild Silkmonths and Economic Insects for Value Added Creation, Khon Kaen University, for their support on the funding and experimental equipments. The appreciation includes Synchrotron Light Research Institute (Public Organization), Thailand for providing the FTIR instruments and beam times.


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Weravart Namanusart [1], Weerasak Saksirirat [1,2] * and Waraporn Tanthanuch [3]

[1] Plant Pathology Section, Department of Plant Science and Agricultural Resources, Faculty of Agriculture, Khon Kaen University - 40002, Thailand.

[2] Agricultural Biotechnology Research Center for Sustainable Economy, Khon Kaen University and Center of Agricultural Biotechnology, Science and Technology Postgraduate Education and Research Development Office (AG-BIO/PERDO-CHE), Thailand.

[3] Synchrotron Light Research Institute (Public Organization), Nakhon Ratchasima 30000, Thailand.

(Received: 07 March 2016; accepted: 30 April 2016)

* To whom all correspondence should be addressed. TeleFax: +66 4334 3114; E-mail:

Caption: Fig. 1. Average raw FTIR spectra from untreated tomato root (control) and treated tomato root with Neonothopanus nambi in different conditions. Spectra were normalized over the range of 3000- 1000 [cm.sup.-1]

Caption: Fig. 2. Representative average 2nd derivative FTIR spectra from untreated tomato root (control) and treated tomato root with Neonothopanus nambi in different conditions. The average spectra were taken to the second derivative to enhance the difference between treated and untreated root which were mainly between 1400-1000 cm1; these include the amide I proteins (1650 [cm.sup.-1]) and 1510 [cm.sup.-1] bands of lignin regions and between 1400-950 [cm.sup.-1] include the poly-saccharide cell wall absorbance regions; the cellulose (1377 [cm.sup.-1]), pectin (1107 [cm.sup.-1]) and hemicelluloses (1060 [cm.sup.-1])

Caption: Fig. 3. Principle component analysis (PCA) of FTIR spectral range 3000- 900 [cm.sup.-1] giving PCA score plots (a) and PCA loading plots (b). PCA score plots showed distinct clustering between untreated (control) root and treated root after 10 days inoculation. PCA loading plots identify biomarker difference (i.e., discriminating wave numbers) over spectral range of samples

Caption: Fig. 4. Cluster analysis of FTIR spectra of untreated tomato root group and root treated group based on spectral range 3000-2800 and 1800-950 [cm.sup.-1]. Classification based on Ward's algorithmn
Table 1. Root galling and number of Meloidogyne incognita J2 in
root 30 days after inoculation on split-root systems of tomato
seedlings with different treatments of Neonothopanus nambi
application on the opposite half-root system at 10 days before
nematode inoculation.

Treatment                      Root galling        Reduction
                                  (%) (b)           (%) (c)

N+ CF PW2/30 ml (a)         4.50 [+ or -] 2.65bc     78.13
N+ SP PW2/ 30 g             3.50 [+ or -] 3.98c      82.99
N+ CF/15 ml+SP/15 g (PW2)   3.50 [+ or -] 3.32c      83.87
N+ Aurisin A /10 ml         5.30 [+ or -] 3.59b      74.25
N+ 0.2% DMSO /10 ml         4.80 [+ or -] 2.50bc     76.68
N-only 3,000 eggs/pot       20.58 [+ or -] 3.86a       -
N-only                             20.6e               -
Control                        0.00 [+ or -]d          -
C.V. (%)                           13.91

Treatment                           No. of            Reduction
                                   J2/g root           (%) (c)

N+ CF PW2/30 ml (a)         138.7 [+ or -] 39.11 e      86.61
N+ SP PW2/ 30 g             170.7 [+ or -] 15.01 d      83.52
N+ CF/15 ml+SP/15 g (PW2)    80.0 [+ or -] 15.10 f      92.28
N+ Aurisin A /10 ml         522.7 [+ or -] 75.72 c      49.55
N+ 0.2% DMSO /10 ml         858.7 [+ or -] 60.58 b      17.11
N-only 3,000 eggs/pot       1036.0 [+ or -] 55.11 a       -
N-only                               0.0e                 -
Control                      0.00 [+ or -] 0.00 g         -
C.V. (%)                                                16.92

Means followed by the same letter (s) are not significantly
different at P>0.05 by DMRT.

(a) Control = no organisms, N = 3,000 M. incognita J2/half-root
system, DMSO=dimethyl sulfoxide, CF = culture filtrate, SP =
mycelial spawn, CF+SP = culture filtrate combined with mycelial

(b) Evaluation in 30 days after inoculation with M. incognita
(3,000 eggs/pot), Treated N. nambi 10 days before inoculation with
M. incognita. Galling index scored based on (25) 0 = no root-knot;
1 = 1-25% root-knot; 2 = 26-50% root-knot; 3 = 51-75% root-knot; 4
= 76-100% root-knot.

(c) Reduction (%) = (Root galling N-only - Root galling
Treatment)/Root galling N-only x 100

Table 2. Contents of phenolic compound, salicylic acid and defense
enzyme activities in the tomato treated root with different conditions
of Neonothopanus nambi at 10 days

Treatment           Phenolic (a)   Salicylic (b)   PAL (c)
                      compound         acid

CF PW2/30 ml           0.093c         0.250ab      17.85b
SP PW2/ 30 g           0.205a         0.268a       29.17a
CF/15 ml + SP/         0.034d         0.265a       31.51a
  15 g (PW2) PW2)
Aurisin a /10 ml       0.128b         0.198c       18.52b
0.2% DMSO/10 ml        0.060d         0.220bc       9.69c
Control                0.050d         0.195c       16.24b
C.V (%)                16.91           18.57        25.99

Treatment           PPO (d)   Chitinase (e)   Protease (f)
                                activity        activity

CF PW2/30 ml         8.51b        3.06c          15.89b
SP PW2/ 30 g        14.15a        5.23a          26.05a
CF/15 ml + SP/       6.48b        2.52d          10.42c
  15 g (PW2) PW2)
Aurisin a /10 ml     6.84b        1.23e          10.72c
0.2% DMSO/10 ml      8.15b        3.93b          10.65c
Control              2.18c        2.97c          18.46b
C.V (%)              21.16        15.69          10.40

Means followed by the same letter (s) in a column are not
significantly different at P>0.05 by DMRT.

(a) Phenolic compound evaluated in subunit [mg [g.sup.-1]
fresh weight];

(b) Salicylic acid [mg [g.sup.-1] fresh weight];

(c) PAL (Phenylalannine ammonia-lyase) [[micro]mol (trans-cinnamic
acid) mg-1 protein/hr];

(d) PPO (Polyphenoloxidase) [[micro]mol (quinone) [mg.sup.-1]

(e) Chitinase activity [[micro]mol (GlcNAc)/[mg.sup.-1] protein)/hr]

(f) Protease activity [[micro]mol (tyrosine)/[mg.sup.-1] protein)/hr]

Table 3. The integral area percentage of cell wall regions assignments
of tomato root untreated and after treated with Neonothopanus
nambi in different conditions

               Cell wall tomato root assignments (integral area %)

Treatment         Protein amide (a)            Lignin (a)
               (1716-1560 [cm.sup.-1])   (1566-1495 [cm.sup.-1])

CF PW2 (1/)      30.8 [+ or -] 0.3ab      0.23 [+ or -] 0.059c
SP PW2           32.8 [+ or -] 1.2a       0.57 [+ or -] 0.058a
CF+SP PW2        25.6 [+ or -] 1.1cd      0.43 [+ or -] 0.000b
Aurisin          22.2 [+ or -] 0.3d       0.30 [+ or -] 0.000bc
DMSO             23.7 [+ or -] 0.5d       0.20 [+ or -] 0.000c
Control          28.2 [+ or -] 0.4bc      0.40 [+ or -] 0.000bc
C.V (%)                 8.14                      5.41

               Cell wall tomato root assignments (integral area %)

Treatment           Cellulose (a)              Pectin (b)
               (1487-1337 [cm.sup.-1])   (1178-1084 [cm.sup.-1])

CF PW2 (1/)      30.6 [+ or -] 0.9b        18.2 [+ or -] 0.1e
SP PW2           18.6 [+ or -] 1.2e        20.2 [+ or -] 3.2d
CF+SP PW2        15.2 [+ or -] 0.7f        25.1 [+ or -] 0.7b
Aurisin          33.2 [+ or -] 1.9a        22.2 [+ or -] 0.2c
DMSO             23.2 [+ or -] 0.5c        31.5 [+ or -] 0.8a
Control          21.0 [+ or -] 0.1d        23.1 [+ or -] 0.5c
C.V (%)                 4.23                      4.28

               Cell wall tomato root
               assignments (integral
                      area %)

Treatment        Hemicellulose (c)
               (1087-975 [cm.sup.-1])

CF PW2 (1/)      20.1 [+ or -] 0.8d
SP PW2           27.8 [+ or -] 1.1b
CF+SP PW2        32.8 [+ or -] 2.5a
Aurisin          22.1 [+ or -] 1.8c
DMSO            21.4 [+ or -] 1.6cd
Control          27.3 [+ or -] 0.6b
C.V (%)                 3.96

Means followed by the same letter (s) in a column are not
significantly different at P30.05 by DMRT

Control = no organisms, DMSO=dimethyl sulfoxide, CF = culture
filtrate, SP = spawn, CF+SP = the combination of

culture filtrate and spawn PW2

(a) from (26), (b) from (27); (28), (c) from (27)
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Author:Namanusart, Weravart; Saksirirat, Weerasak; Tanthanuch, Waraporn
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Jun 1, 2016
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