Evaluation of microbial elicitors to induce plant immunity for tomato wilt

1 Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Ladkrabang, Bangkok, 10520, Thailand. 2 Department of Agriculture, Faculty of Science and Technology, Nakhonratchsima Rajabhat University, Thailand. 3 Natural Products Research Unit, Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand.


INTRODUCTION
Plants possess a broad spectrum of basic defense mechanisms, pre-established or induced secondary metabolites with antimicrobial activities, which render them resistant to most potential colonizers.Many of these com-pounds are presented in healthy plants as normal metabolic products, whereas, in other cases, they can be actively syn-thesized in response to pathogen attack (Roncero et al., 2003).Elicitors are molecules that stimulate any of a number of defense responses in plants, such as synthesis of phytoalexins and pathogenesis-related proteins (PR-proteins).Such responses occur after the binding of elicitor molecules to receptors normally located on the plant cell surface, promoting a signal transduction pathway that leads to the activation of one or more defense mechanisms.The first characterized elicitors were oligosaccharide fragments from fungal cell walls, including hepta-β-glucoside, oligochitin and oligochitosan (Hahn, 1996).VanEtten et al. (1994) mentioned that the definition of antibiotic compounds produced in the part of plant as phytoalexins are low molecular weight compounds which are both synthesized by and accumulated in plants after exposure to microorganisms and antifungal compounds may be presented constitutively in one part of a plant but induced as phytoalexins in other organs.Morrissey and Osbourn (1999) stated that phytoalexins are a group of structurally diverse molecules that are generally lipophilic, nonspecific in their antifungal activity, and not particularly potent.The accumulation of phytoalexins represents one of an array of induced defense responses associated with plant disease resistance.Although both disease-resistant and susceptible plants may respond to pathogen attack by producing phytoalexins; these compounds generally accumulate more rapidly and to higher levels in resistant plant.With this, Roldán et al. (1999) reported that plants have evolved different defense mechanisms to protect themselves against a great variety of invasive pathogens.Moreover, Roddick (1974) studied a determinant of resistance of tomato plants to fungi present in the plant of a preformed inhibitor of fungal growth namely α-tomatine.The α-tomatine is a glycosidal alkaloid consisting of an aglycone moiety (tomatidine) and a tetrasaccharide moiety (β-lycotetraose) which is composed of two molecules of glucose and one each of galactose and xylose; the four monosaccharides form a branched structure which is attached at the C-3 position of the aglycone.It has been calculated as C 50 H 83 NO 21.Moco et al. (2006) and Sandrock and VanEtten (1998) reported that α-tomatine expresses a molecular weight (MW) = 1,033.5458and Rf = 0.23.However, Roddick (1974) studied on the method for quantitative estimation of tomatine involves in spectrophotometry of a chromogen of α-tomatine showing coloured products formed by treating tomatine with strong or concentrated H 2 SO 4 , a lactic acetic solution of silicotungstic acid and α-tomatine found at high concentrations (up to 1 mM) in leaves, stems, roots, and green fruit.He reported that the shoot is recognized as being the main site of α-tomatine synthesis it accumulates in the meristematic region, roots, flowers and young developing fruits but fruits ripening begins alkaloid degradation occurs and its concentration declines.Then, the αtomatine levels of 0.087, 0.045 and 0.036% have been recorded in green, yellowish and red tomato fruit respectively, and when ripe fruits were left on the plant for further 2-3 days, α-tomatine almost completely disappeared.Lairini et al. (1996) reported that the -tomatine presents in tomato plants as the antifungal compound, has been reported to provide a preformed chemical barrier against phytopathogenic fungi and some fungi are resistant to -tomatine because of their membrane composition, while others produce specific tomatine-detoxifying enzymes as F. oxysporum f. sp.Lycopersici; a tomato wilt causing agent, which produces an extracellular enzyme inducible by -tomatine.This enzyme, known as tomatinase, catalyzes the hydrolysis of -tomatine into its nonfungitoxic forms, tomatidine and β-lycotetraose.Then, tomatinase remove all four sugars from the steroidal tomatine and an inducible enzymatic activity of F. oxysporum f. sp.lycopersici was able to detoxify -tomatine by cleaving the glycoalkaloid into the tetrasaccharide lycotetraose and tomatidine.Friedman (2004) stated that the methods used to analyze -tomatine include Gas chromatography (GC), Gas chromatography-mass spectrometry (GC-MS), and high performance liquid chromatography (HPLC).With this, the -tomatine presents in all parts of the tomato plant.Immature green tomatoes contain up to 500 mg/kg of fresh fruit weight.Then, consumers of green tomatoes, high -tomatine red tomatoes, and tomato products such as pickled green and green fried tomatoes consume significant amounts of -tomatine.Finally, the tomatine has been determined in tomato by HPLC using pulsed amperometric detection.Friedman and Levin (1995) detected -tomatine in different parts of the tomato plant using pulsed amperometric detection HPLC.The results demonstrated that the quantity of -tomatine from calyxes, flowers, leaves, roots, and stems of the tomato plant were detected as 14-130 mg/100 g of fresh weight.Red tomatoes contained low-tomatine as 0.03-0.08mg/100 g.The intermediate stage of tomato fruit contained -tomatine between 0.1-0.8mg/100 g plant fresh weight.High quantity of -tomatine was detected from fresh green fruits as 0.9-55 mg/100 g plant fresh weight.Moreover, Melton et al. (1998) reported that tomato seedlings var.Moneymaker Cf4 and Cf5at 11 day-old were inoculated with conidial suspension of Cladosporium fulvum at concentration as 5 × 10 5 conidia/ml.Result reveal that the -tomatine were detected from overground part of tomato seedlings and the contents of -tomatine in 11-day-old seedlings var.Moneymaker Cf4 and Cf5 range from 260 to 380 and from 260 to 579 g/g of fresh weight, respectively while the tomatine contents of the leaves of 3-week-old plants var.Moneymaker Cf4 and Cf5 were 750 to 1,260 and 730 to 850 g/g of fresh weight, respectively.The objective of this study was to elucidate the bioactive compounds of Chaetoglobosin C, chaetomanone A and trichotoxin mixture used as microbial elicitors to elicit -tomatine in tomato.

Microbial elicitors inducing plant immunity
Chaetoglobosin C from Chaetomium globosum, white or cream solid, chaetomanone A from Chaetomium lucknowense, yellow crystal, and trichotoxin mixture from Trichoderma harzianum, white solid, were used as microbial elicitor to induce plant immunity against F. oxysporum f.sp.lycopersici causing tomato wilt.The experiment was per-performed to detect α-tomatine in laboratory.The trial was conducted with a modified method of Melton et al. (1998) and designed in Completely Randomized Design (CRD) with six treatments and three replications.Twenty-day-old seedlings var.Sida were inoculated with conidial suspension of F. oxysporum f. sp.lycopersici at concentration of 2×10 6 conidia/ml and planted into 4 inch diameters plastic pots containing sterilized soil mixture and followed by spraying each bioactive compound or chemical fungicide on the seedling leaves.Treatments were as follows: inoculated with pathogen and sprayed with Chaetoglobosin C at 50 g/ml (T1), inoculated with pathogen and sprayed with Chaetomanone A at 50 g/ml (T2), inoculated with pathogen and sprayed with trichotoxin mixture at 50 g/ml (T3), inoculated with pathogen and sprayed with prochloraz at 20 g/20 l of water (T4), only inoculated with pathogen (T5) and non-inoculated control (T6).Disease severity index (DSI) was determined before seedlings were harvested for detection of α-tomatine quantification.Percentage of plant disease immunity (PDI) on treated tomato seedlings were analyzed using the formula: DSI of (inoculated control -DSI of each treatment / DSI of inoculated control) x 100.The α-tomatine was extracted from 6 g of stems and leaves of tomato harvested at 5, 10 and 15 days after treatment.Then plant tissues were ground in 95% methanol with a pestle and mortar.The extract was evaporated in vacuum evaporator and adjusted with methanol and then they were applied to thin layer chromatography (TLC) with standard comparison of α-tomatine.The spot of extracts from tested tomato which showed Rf = 0.23 (solvent system eg.30% ethyl acetate-hexane) as α-tomatine was expected to be α-tomatine of each treatment.The α-tomatine quantification of the extracts was performed by using HPLC (high performance liquid chromatography) with modified method of Friedman et al. (1994).The crude extracts from tested plants were weighted (1 mg) and were dissolved with 1 ml of mixed solvent comprised of 50% methanol -0.1% acetic acid and filtered through 0.45 µm nylon membrane.The filtrate at 50 µl was subjected into HPLC (Agilent Co. ltd.).The HPLC eluent for αtomatine analysis was prepared by combining solvent system with HPLC grade of 80% water: 15% acetonitride (CH3CN): 5% methanol.Flow rate was set to 1.0 ml/min.The C8 chromatography column was eluted with HPLC solvent system before the filtrates were injected.The α-tomatine quantification was done using linear regression curve.Data was statistically computed by analysis of variance.Treatment means were compared with Duncan's New Multiple Range Test (DMRT) at P= 0.05 and P= 0.01.

Evaluation of microbial elicitors to control tomato wilt
The second experiment was done by using Randomized Completely Block Design (RCBD) with four replications and treatments were designed as follows: inoculated seedling roots of tomato treated with chaetoglobosin C at 10 µg/ml (T1), at 50 µg/ml (T2) and at 100 µg/ml (T3), treated with chaetomanone A at 10 µg/ml (T4), at 50 µg/ml (T5) and at 100 µg/ml (T6), and treated with trichotoxin mixture at 10 µg/ml (T7),at 50 µg/ml (T8) and at 100 µg/ml (T9), treated with chemical fungicide (prochoraz 50% WP) at 20 g/20 l of water (T10), and inoculated (T11) and non-inoculated controls Tomato seedlings var.Sida at 30 days old were inoculated with the most aggressive isolate of F. oxysporum f. sp.lycopersici at concentration of 2x10 6 conidia/ml using dipped root method which was modified from Sibounnavong et al (2009).Inoculated seedlings were transferred into 11 inch plastic pot which contained sterilized mix soil (soil: sand: compost, 4:1:1).Each experimental unit was planted to 8 seedlings of tomato.The bioactive compounds in each concentration and prochloraz were Kasem et al. 1995 sprayed over tomato leaves immediately and every two weeks after transplanted.Data were collected as growth parameters every 15 days; such as plant height (cm) and diameter of plant canopy (cm).Disease severity index (DSI) was scaled as previous experiment and percentage of plant disease immunity (PDI) was analyzed.Data were subjected to analysis of variance (ANOVA) and treatment mean was compared with DMRT at P=0.05 and P=0.01.Disease reduction and % increase in yield were computed using the formula as previous shown.

RESULT Microbial elicitors inducing plant immunity
α-Tomatine was induced by bioactive compounds such as chaetoglobosin C, chaetomanone A and trichotoxin mixture.
Results show that PDI on treated tomato seedlings with chaetoglobosin C, chaetomanone A and trichotoxin mixture after 15 days were 44.97, 35.18 and 39.43%, respectively when compared to prochloraz that showed PDI of 29.95% (Table 1).The extracts from tested tomato were spotted to thin layer TLC paper with standard comparison of αtomatine.The spot of extracts on TLC paper presented green spot in all treatments with Rf = 0.23 as same as standard α-tomatine (Figure 1).The quantity of α-tomatine at five days were analyzed and the data revealed that inoculated seedlings followed by treatment with chaetoglobosin C showed the highest α-tomatine quantity as 746.67 ug/g of sample while inoculated seedlings followed by treatment with chaetomanone A, and trichotoxin mixture showed significantly lower α-tomatine quantity as 535.01 and 599.70 ug/g of sample.Meanwhile, inoculated seedlings followed by treatment with prochloraz, inoculated control, and non-inoculated control showed non-significant difference in α-tomatine quantity as 368.68, 361.51, and 294.38 ug/g of sample, respectively.The treatment with trichotoxin mixture showed α-tomatine quantity as 492.22 ug/g of sample which had higher significant different than treatments with chaetoglobosin C, chaetomanone A, and non-inoculated control which showed α-tomatine quantity as 348.72, 322.98, and 321.19 ug/g of sample after ten days.In addition, inoculated seedlings followed by treatment with prochloraz and the inoculated control showed lowest quantity of α-tomatine as 190.82 and 179.87 ug/g of sample.When compared with 5 days and 10 days, the quantity of α-tomatine at 15 days showed fewer amounts in all treatments except for the non-inoculated control which revealed the content of α-tomatine as 365.91 ug/g of sample.Treatments with chaetoglobosin c, chaetomanone A, and trichotoxin mixture showed not significant difference in quantity of α-tomatine as 207.87, 254.25, and 205.04 ug/g of sample, respectively while treatment with α-tomatine were 131.56 and 77.46 ug/g of samples, respectively.

Evaluation of microbial elicitors to control tomato wilt
The bioactive compounds, chaetoglobosin C, chaeto-    manone A and trichotoxin mixture were determined for the efficacies as microbial elicitors to induce plant immunity for tomato wilt in vivo (Table 2).At 30 days after treatments, disease severity index (DSI) of inoculated control showed the highest DSI of 3.88 while prochloraz treatment showed lower DSI of 3.0.The treatment of all concentrations of bioactive compounds presented the same level of DSI ranging from 1.50 to 2.25.Plant disease immunity (PDI) of treatment with trichotoxin mixture at 50 µg/ml revealed highest plant disease immunity of 63.96%.Treatments with bioactive compounds of chaetoglobosin C at 10, 50 and 100 µg/ml, chaetomanone A at 10, 50 and 100 µg/ml, and trichotoxin mixture at 10 and 100 µg/ml showed lower plant disease immunity than 50 µg/ml of trichotoxin treatment while prochloraz treatment showed the lowest plant disease immunity of 21.94%.Result shows that tomato treated with chaetoglobosin C at 10, 50 and 100 µg/ml, chaetomanone A at 10 and 50 µg/ml, trichotoxin mixture at 10 µg/ml and the non-inoculated control revealed similar plant height (Table 3).The treatments of chaetomanone A at 100 µg/ml, trichotoxin mixture at 50 and 100 µg/ml, prochloraz and the inoculated control showed lower significantly plant height.Plant canopy of tomatoes treated with chaetoglobosin C at 100 µg/ml and chaetomanone A at 50 µg/ml showed the highest result at 31.13 and 31.50 cm, respectively.Tomatoes treated with chaetoglobosin C at 10 and 50 µg/ml, chaetomanone A at 10 and 100 µg/ml, trichotoxin mixture at 10 and 50 µg/ml and non-inoculated control showed non-significant canopy while tomatoes treated with prochloraz and the inoculated control showed lower significant plant canopy as compared with other treated tomato plants.
The results of the experiment at 45 days after treatments show that the inoculated control showed the highest disease severity index (DSI) of 4.25 while prochloraz treatment showed lower significantly DSI of 3.13.The treatments with chaetoglobosin C at 10 µg/ml resulted in significantly higher DSI than for other treatments of bioactive compounds while chaetoglobosin C at 50 and 100 µg/ml, chaetomanone A at 10, 50 and 100 µg/ml, trichotoxin mixture at 10, 50 and 100 µg/ml showed significantly lower DSI as compared with 10 µg/ml of chaetoglobosin C treatment.PDI of trichotoxin mixture at 50 µg/ml revealed the highest plant disease immunity of 65.15% while the treatment with chaetoglobosin C at 10, 50 and 100 µg/ml, chaetomanone A at 10, 50 and 100 µg/ml and trichotoxin mixture at 10 and 100 µg/ml showed lower plant disease immunity.The treatment of prochloraz showed significantly lower plant disease immunity than bioactive compounds (Table 2).Result of plant height show that tomato treated with bioactive compounds in all concentrations and the non-inoculated control pre-sented non-significant plant height (Table 3).Plant canopy of treatments with chaetoglobosin C at 100 µg/ml, chaetomanone A at 50 µg/ml and the non-inoculated control showed significantly higher plant canopy when compared to treatments of trichotoxin mixture at 10 and 50 µg/ml while treatments with chaetoglobosin C at 10 and 50 µg/ml, chaetomanone A at 10 and100 µg/ml and trichotoxin mixture at 100 µg/ml showed sig-nificantly higher plant canopy than prochloraz treatment and the inoculated control.
The results of 60 days after treatments revealed that disease severity index and plant disease immunity index of all treatments presented the same results as the treatments at 45 days (Table 2).Plant height of all concentrations of bioactive compounds and the non-inoculated control presented no significant difference (Table 3) while prochloraz treatment and the inoculated control showed significantly lower plant height.The results of plant canopy show that tomatoes treated with chaetomanone A at 50 µg/ml and trichotoxin mixture at 10 µg/ml showed significantly higher plant canopy than for other treatments.Treatments with chaetoglobosin C at 10, 50 and 100 µg/ml, chaetomanone A at 10 and 100 µg/ml, trichotoxin mixture at 50 and 100 µg/ml showed no significant difference with the non-inoculated control.Plant canopy of tomatoes treated with prochloraz and the inoculated control showed significantly lower plant canopy.The results of the treatments are demonstrated in Tables 2 and 3.

DISCUSSION
α-Tomatine was induced by chaetoglobosin C from Chaetomium globosum, chaetomanone A from C. lucknowense and trichotoxin mixture from T. harzianum when treated as microbial elicitor to inoculated tomato seedlings with F. oxysporum f.sp.lycopersici causing tomato wilt.The disease immunity on treated tomato seedlings with chaetoglobosin c, chaetomanone A and trichotoxin mixture after 15 days were 44.97, 35.18 and 39.43%, respectively when compared to prochloraz that showed PDI of 29.95%.The spot of plant extracts on TLC paper presented green spot in all treatments with Rf = 0.23; same as standard α -tomatine (Friedman, 2004).The quantity of αtomatine presented in all parts of tomato plant at ten days showed that trichotoxin mixture induced α-tomatine quantity (492.22 ug/g) followed by treatment with chaetoglobosin C and chaetomanone A (348.72 and 322.98, ug/g).Friedman (2004) stated that -tomatine is present in all parts of the tomato plant.Immature green tomatoes contain up to 500 mg/kg of fresh fruit weight.As Soytong et al. (2001) reported, trichotoxin mixture and chaetoglobosin C can actively act as elicitor to induce phytoalexin in tomato and potato plants and applied as elicitors to induce oxidative burst in may plants as the first signal for plant immunity.
The research finding exhibited bioactive compounds; chaetoglobosin C from Chaetomium globosum, chaetoma-manone A from Chaetomium lucknowense and trichotoxin mixture from T. harzianum expressed a strong inhibitory activity in vivo against tomato Fusarium wilt.With this, inoculated tomato plant applied with bioactive compounds at 10-100 µg/ml showed higher plant disease immunity (53.80-65.15%)than prochloraz (26.73%).This result was supported by the report of Soytong et al. (2001) and Suwan et al. (2000) who stated that chaetoglobosin C and trichotoxin mixture could elicit resistant or immunity in tomato and other plants, inhibit the pathogen and stimulate plant growth.With the results, tomato plants treated with bioactive compounds showed significant difference in plant height, plant fresh and dry weight than chemical prochoraz and inoculated control.This was similar as Soytong et al. (2007) reported that trichotoxin mixture exhibited a potential as plant growth regulators in Chinese cabbage, kale and mungbean.There are similar reports which indicated that bioactive compounds from C. globosum and T. harzianum gave a good control of plant pathogenic fungi but different found compounds such as chaetoviridins A extracted from C. globosum F0142 could inhibit the growth of Magnaporthe grisea and Phytophthora ultimum with MIC value of 1.23 µg/ml (Park et al., 2005); chaetomin from liquid culture of C. globosum exhibited activity against damping off of sugar beet caused by Pythium ultimum (Di Pietro et al., 1992), and antifungal metabolite production by C. globosum exhibited the ability to control spot blotch of wheat caused by Cochliobolus sativus (Aggarwal et al., 2004).Viridiofungin A produced from T. harzianum isolate T23 retarded the mycelial growth of F. moniliforme (El-Hasan et al., 2009).Srinon et al. (2006) and Sibounnavong et al. (2009Sibounnavong et al. ( , 2012) ) stated that F. oxysporum f. sp.lycopersici causing tomato wilt was also inhibited by the crude extract from Emericella nidulans and Emericella rugulosa.This was to confirm that bioactive compounds from several antagonistic fungi could suppress and feasibly act as microbial elicitors to induce plant immunity.
This research finding demonstrates that chaetomanone A was a known compound but was firstly discovered from C. lucknowense.Previous report of Kanokmedhakul et al. (2002) stated that chaetomanone A was firstly extracted from C. globosum strain N0802 and it also exhibited antitubercular activity against Mycobacterium tuberculosis, a major human disease.But there are no reports on chaetomanone A against plant pathogenic fungi.It is suggested that different strains of C. globosum, C. lucknowense and T. harzianum which could differ in antibiotic production implies specific antibiotic production strains.This result suggests that chaetoglobosin C, chaetomanone A and trichotoxin mixture play an important role as microbial and also in antagonism and antibiosis against F. oxysporum f. sp.lycopersici causing tomato wilt.Kasem et al. 1999 As the results of research, C. globosum KMITL-N0802, C. lucknowense CLT and T. harzianum PC01 and their antagonistic substances including their bioactive compounds, chaetoglobosin C, chaetomanone A and trochotoxin A50 are effective biological control agents which act in multi mechanism of actions to control Fusarium wilt.Their potential include eliciting of immunity in tomato plants, production of antibiotic substances to inhibit growth of pathogen, decrease of disease incidence and also stimulatation of plant growth.However, Sibounnavong et al. (2012) firstly reported that antibiotic substance, chaetoglobocin C from C. lucknowense controlled F. oxysporum f. sp.lycopersici causing tomato wilt and this compound had greater antifungal activity against Fusarium wilt pathogen which implies antibiosis as control mechanism for plant disease.The formulation of fungal products should be further developed as biofungicides for safety agriculture.

1
Average of three replications.Means with the same common letters in each column are not significantly different according to Duncan's multiple range test at P = 0.01; 2 Plant disease immunity (PDI) = (DSI of inoculated control -DSI of each treatment) / DSI of inoculated control x 100.

Table 1 .
Disease severity index and α-tomatine quantification of tomato after inoculated with pathogen and sprayed with bioactive compounds.

Table 2 .
Disease severity index and plant disease immunity of tomato at 30, 45 and 60 days after treatments.

Table 3 .
Plant growth parameters of tomato at 30, 45 and 60 days after treatments.