Marigold as an Effective Candidate in Nematode Control

Table of contents

Preface

Introduction

Methodology

Results of the studies

Discussion

Literatures Cited

Preface

Nematodes are tiny, microscopic round worms which are subterranean in nature and affect almost all the agricultural crops and cause substantial yield loss to the farmers. Among them, root knot nematode, Meloidogyne incognita is a polyphagous pest and affects all the vegetable crops. This nematode along with pathogenic fungi and bacteria aggravate the disease severity and ultimately leads to the death of the plants.

Nematode management over years relied on chemical methods. Owing to the ill effects with the application of chemicals, ecofriendly methods are gaining importance now a days in nematode management.

Marigold is an effective antagonistic plant against nematodes. The plant, by the production of alpha terthienyl bithenyl compounds from its roots, repel the nematodes and reduce its population. Understanding the mechanism involved in the antagonistic nature of the plants will result in the better enhancement of the nematode management practices in an environmental friendly approach.

K. Sankari Meena

M. Sivakumar

Marigold, an effective candidate in nematode control

Introduction

Tomato (Lycopersicon esculentum Mill .) is the most important solanaceous vegetable. In India, it is produced around 1, 75, 00,000 MT/ha. Lycopene, an antioxidant is the primary product of tomato, which gives colour to the fruit and protects humans against cancer and heart diseases. Moreover the fruit is also rich in Vitamin A and C.

Among the yield reducing factors in tomato, root knot nematode, Meloidogyne incognita is the only species known to cause economic damage and often happens to be the major limiting factor in the successful cultivation of tomato (Lycopersicon esculentum Mill.) In affected roots, the functional root system gets modified to galls and it impairs the uptake of water and nutrients. Poor development of root system makes the plant highly susceptible to drought. The entire root system may be shallow with excessive branching.

Annual yield loss due to this nematode is 49 per cent (Zalom, 2003). Use of chemical nematicides is gradually phased out worldwide due to their high mammalian toxicity and alternative methods are being taken up. Now a days botanicals are gaining importance because of their easy availability, low cost and ecofriendly nature. Many workers have studied the efficacy of the botanicals against nematode species with varying degrees of success.

Marigold (Tagetes spp. ,) is an excellent candidate for the management of root knot nematodes; the chemicals present in their root system of the plant effectively check the nematode population. Use of Marigold in nematode control is an environmentally safer and economically viable method.

According to Siddiqui and Alam (1988), aerial parts of Tagetes lucida was found to be more toxic than their roots. Tagetes erecta suppressed the galls caused by M. javanica in tomato. T. erecta and T. patula were able to suppress the population of Meloidogyne, Radopholus and Pratylenchus in banana. Vegetative stage of T. erecta however was longer than T. patula, so its effective age for controlling plant parasitic nematodes on banana was also longer than T. patula (Visser and Vythilingam, 1958; Supratoyo, 1993).

Nematicidal property of root exudates of T.minuta, Cannabis sativa and Aloe barbidensis against root knot nematode species has been reported by Wani and Ansari (1993); Singh and Veena Singh (2002). Organic matter obtained from T. minuta; Ricinus communis and Datura stramonium was found to stimulate the parasitism of M. incognita eggs by Paecilomyces lilacinus fungi (Odour-Owino et al., 1993). Nahar et al. (1996) stated that root knot nematode in tomato can be suppressed by the mixed application of poultry manure, mustard oil cake, pigeon manure and Tagetes species. Similarly, higher mortality rate of juveniles of M. incognita was observed with the application of marigold (T. erecta) and garlic (Ali et al.,1995). Mojumder (1998) also found that growing Tagetes species could control the juveniles of M. incognita.

Azadirachta indica and T. patula were effective in improving the growth of tomato plant by reducing the nematode population and gall index (Khanna and Sharma, 1998). Similarly, M.incognita on Capsicum annum was effectively controlled by T. patula under laboratory conditions (Mareggiani et al., 1997). Verma and Ali Anwar (1998) found the highest toxic effect of Marigold cv. Saffron Spice on the gall formation on pointed gourd (Trichosanthes diocia).

Nematicidal activity of the flowers of T. erecta was found to be effective against M. incognita, M. javanica (Madhulu et al., 1994 ) and Rotylenchulus reniformis (Rakesh Pandey et al., 2001). Dhangar et al. (2002) identified the nematicidal property of root exudates of T. minuta against M. incognita. Mortality rate of M. incognita increased with increase in concentration and exposure period of root exudates. Marigold should be grown at soil temperature above 15[0] C to suppress M. incognita infection on the subsequent crop (Ploeg and Maris, 1999).

Growing Tagetes as intercrop for nematode control

Under field condition, cropping of T. erecta and incorporation of its residues resulted in significant reduction of M. incognita population in tomato (Akhtar and Alam, 1992; Ploeg, 2000; Sellami and Zemmouri, 2001).

T. erecta, when grown as intercrop with different varieties of tomato during winter and different varieties of okra during summer brought reduction in the population of the species of Meloidogyne, Tylenchorhynchus, Helicotylenchus, Hoplolaimus, Rotylenchulus and Pratylenchus (Khan et al.,1971). It was also found that it is a poor host for stunt nematode, Tylenchorhynchus brevilineatus and reduced nematode population in soil and pod disease severity and increased groundnut pod yield (Naidu et al., 2000) when intercropped with groundnut.

Nematode population greatly decreased in tomato at 90 or 120 days after the cultivation of T. patula and crop yield increased upto 50 per cent (Sano et al., 1983; Ploeg, 2002). Omidvar (1962) observed that potato yield from the plots either formerly planted with marigold or left fallow were significantly greater than those from the plots formerly planted with potatoes, thus marigold was found to be effective in reducing lesion nematode populations in potato soil (Alexander and Waldenmaier, 1999; Kantharaju and Reddy, 2001). Root knot nematode disease in the eggplant was controlled by growing Tagetes in alternate as well as alternatively in the same row (Daulton and Curtis, 1963; Varma et al., 1978).

T. patula and T. erecta along with brinjal cv. Pusa Kranthi in the field recorded a significant reduction in the population of M. incognita and Helicotylenchus indicus. Banana plants when intercropped with T. erecta, Coriandrum sativum and Crotalaria juncea, greater reduction in M. incognita, Helicotylenchus multicinctus and Hoplolaimus indicus was observed (Nagananthan et al., 1988; Charles, 1995). Tomato plants when grown with T. minuta had significantly greater shoot growth and fruit yield than the untreated plants and also the intercrop controlled the population of M. javanica (Abid and Maqbool,1990; Odour-Owino, 1993; Yen et al., 1998). Thus, it was found that growing Marigold (T. patula, T. erecta and T. minuta) as intercrop reduced the population of nematodes as effective as non host plants (Toida et al., 1991; Kimpinski and Arsenault, 1994).

Feasibility of using marigold (T. patula) during the pineapple inter cycle periods was investigated by Prasad et al. (1992) and found that marigold reduced the population of Rotylenchulus reniformis and Pratylenchus spp. and also reduced Pratylenchus penetrans in apple when it is intercropped (Ko and Schmitt, 1993; Edwards et al., 1994). Casewell et al. (1991) found that T. patula decreased the population of P. penetrans resulting in an increased bulb yield of narcissus and lily. T. patula intercropped with chrysanthemum controlled the population of M. incognita, P. penetrans and R. reniformis ( Ramakrishnan et al., 1995).

Prasad and Haque (1982) reported that when garlic and T. erecta were planted alone or combined with infected tomato plant, T. erecta was more effective in suppressing the root knot galls (Alexander and Waldenmaier, 2000) when compared to garlic . Koot and Kroohen-Backbier (1999) advocated the cultivation of T. patula before the cultivation of a susceptible crop for the control of root knot nematode. There was a reduction of Meloidogyne spp. in the soil and the roots of the plants interplanted with T. patula ( Mauch and Ferraz, 1996).

Reynolds et al. (2000) rotated T. patula cv. Creole and T. erecta cv. Crackerjack with traditional rye crop and also applied chemical fumigation prior to transplanting flue cured tobacco and observed that within 75 days of seeding marigold, P. penetrans population was reduced to less than 100/kg soil, far below the ETL of 500 nematodes/kg soil. A plant density of about 20 plants/m[2] reduced P. penetrans population densities to levels below ETL for the rotation crop year and the following years. Tobacco yield was increased by a mean of 197 kg/ha by marigold rotation crops compared with rye and chemical fumigation.

Decomposition of marigold proceeds normally without leaving any toxic decomposers (Ball Coelho et al., 2001). A higher top soil nitrate concentration was also observed under marigold cultivation (1.1 mg/gm) and it was concluded that marigold rotation may be a viable alternative to rye, but to minimize nitrogen loss, marigold crops should be left standing over winter and preplant fertilized with 45 kg nitrogen/ha.

Extracts of Tagetes in nematode control

Root dip treatment of eggplant seedlings with marigold leaf extracts reduced the development of M. incognita (Hussaini et al., 1984). Similar effect was observed with the foliar and soil application of the extract. Extracts of T. erecta showed good nematicidal activity against M. arenaria, M. hapla and M. javanica but not on M. incognita.

Methanolic leaf extracts of T. patula at 1:5 dilutions acheived 75 to 100 per cent mortality against Tylenchulus semipenetrans and Anguina tritici (Mojumder and Misra, 1991). T. patula leaf extracts at dilutions of 1:1, 1:5, 1:10, 1:20, 1:50 and 1:100 were found to be effective against Radopholus similis on banana (Subramanian and Selvaraj,1988). All the nematodes were killed or inactivated after 4h in 1:1 and 1:5 dilutions. The same effect was seen after 48h in 1:10 and 1:20 dilutions. At 1:50 and 1:100 dilutions, the nematicidal effect was found to be significantly reduced. Egg hatching was inversely proportional to the concentration of the extract and directly proportional to the exposure period (Joymathi et al., 1999).

Root extracts of T. erecta were tried in vitro against M. incognita and found to exhibit nematicidal activity (Sharma and Thrivedi,1992; Sasanelli and Addabbo, 1992; Dhangar et al., 1996). Leaf extracts of T. patula at 1:5, 1:10 and 1:20 dilutions were found to be significant in reducing the galls and egg mass production (Mateeva and Ivanova, 2000; Khan and Siddiqui, 2001).

Hatching of M. incognita was inhibited considerably when treated with aqueous extracts of 30 and 60 days old T. patula plants. The juveniles hatched in 60-day-old plant extracts were immobilised (Mateeva, 1995). Hussaini et al. (1984) reported the effectiveness of leaf extracts of T. erecta at 1:2 and 1:4 dilutions in inhibiting the egg hatch of M. javanica (Hussaini et al., 1997), M. arenaria (Sosamma and Jayasree, 2002) and M. incognita in blackgram (Sankaranarayanan and Sundarababu, 1996; Upadhyay et al., 2003). The treatments also significantly increased the shoot weight, root weight and the fruit yield of tomato (Saravanapriya, 2002).

Water extracts of different parts of T. lucida were highly deleterious to M. incognita, R. reniformis, Tylenchorhynchus brassicae and H. indicus to varying degrees (Mansoor et al., 1988). The nematode mortality increased with the increase in the concentration of the extract and exposure period. Flower extracts of T. lucida caused greater mortality and also inhibited the juvenile hatching followed by seed, leaf and root extracts. Nematicidal activity of methanol extracts of T. erecta was more when compared to T. patula ( Zavaleta - Mejia and Gomez, 1995).

Air dried and finely powdered roots of T. erecta, T. patula and T. minuta which were extracted with petroleum ether and chloroform were highly potent against nematodes and T. erecta extracts exhibited more nematicidal effect against M. incognita than extracts from other species (Gengaihi et al.,2001; Cannayane and Rajendran, 2002).

Dry powder of Tagetes on nematode mortality

Leaf powder of T. erecta at 5, 10 and 15 g / kg of soil recorded significant reduction in the reniform nematode population in soybean and also caused phytotoxicity to soybean when added at 15 g (Al Sayed et al., 1992). Meloidogyne and Hoplolaimus populations were reduced by the application of dry leaf powder of T. erecta (Sharma and Thrivedi, 1995). Dried, chopped portion of rice, marigold (T. erecta) and wheat at the rate of 10, 20, 30g/pot caused reduction in the egg mass production and gall formation in sunflower plants infected with M. incognita (Abadir et al., 1994).

Application of dried, chopped leaves of marigold at 500, 1000 and 1500g/m[2] gave the highest reduction in galls and egg masses of M. incognita and M. javanica on tomato, Other nematodes viz., Helicotylenchus sp., Hoplolaimus sp., Tylenchorhynchus and Tylenchulus were also controlled significantly (Rangaswamy and Reddy,1993). Incorporation of chopped Tagetes leaves at 40 and 80 g/kg of soil and recorded a significant reduction in number of galls, egg masses and final population of M. javanica in soil and also increase in the plant growth parameters in tomato and brinjal (Kum Kum Walia and Gupta, 1997). Dry powder of T. patula and T. erecta significantly reduced egg hatching and survival of M. incognita (Yen et al., 2000).

Compatibility of Tagetes with chemicals in the suppression of nematodes

Root dip treatment of eggplant seedlings with margosa, marigold leaf extracts, aldicarb, mustard cake and carbofuran reduced the development of M. incognita compared to chenopodium leaf and groundnut cake treatments (Hussaini et al., 1984).

Under field conditions, in Piper betle, highest reduction in galls on roots was observed with aldicarb treatment followed by neem cake and T. erecta (Rodriguez - Kabana et al., 1988). Combination of marigold with vermiculite recoded highest plant growth and minimum galls and egg masses. Captafol along with the leaf extracts of T.minuta and D. stramonium significantly inhibited the fungal parasitism of M. incognita and M. javanica eggs (Owino, 1992; Makhatsa et al., 1993).

In tomato, under pot culture condition, better plant performance with low gall index was observed in the plants treated with Paecilomyces lilacinus in combination with aldicarb and T. minuta (Odour- Owino et al., 1996). Pratylenchus on strawberry was controlled by the combined effect of fumigation with dazomet and Tagetes (Faby, 1997).

Bioactive principles of Tagetes

Zechmeister and Land Sease (1947) isolated a blue fluorescing compound, α - terthienyl (C 12H 8 S3) from the petals of the common marigold (T. erecta) flower . The compound has been isolated as pure crystals. The nematode suppressing properties of Tagetes spp. in vivo are due to α -terthienyl, 5 - (3-buten - 1-ynyl)- 2 and 2' -bithienyl as such in the roots of the plants, which were effective against Globodera and Pratylenchus (Uhlenbroek and Bijloo, 1959).

It is more likely that in vivo energy rich related forms are the active agents and they show more nematicidal property near UV light than in dark (Gommers, 1972; Jaap Bakker et al., 1979). Gupta and Bhandari (1974) studied the chemical composition of T. erecta flowers and found to contain limonene (31.4 %), ocimene (18 %), linalool (13.6 %), α - d - phellandrene (3.5 %), linalyl acetate (11.6 %), tangetone (4 %), n - nonylaldehyde (6 %), and 1:8 cincelol (11.9 %).

Non-volatile chemical constituents of Tagetes has lutein acetylated with fatty acids (Philip and Berry, 1975; Piccaglia et al., 1998). T. minuta possess acyclic, monocyclic and bicyclic monoterpenes, sesquiterpenes, flavanoids, thiophenes and aromatics (Rodriguez and Mabry, 1977) and these compounds are the effective deterrents of nematodes (Grainge and Ahmed, 1988). Baslas and Singh (1980) extracted yellow essential oils by steam distillation from the flowers of T. erecta and identified the compounds α - pinene, β - pinene, dipentine, menthol, geraniol along with d - limonene, tagetone, linolool, 1:8cineole and linalyl acetate. Thiophene and α - terthienyl in the roots of Tagetes were effective against M. incognita (Castro and Munaz, 1982; Gau et al., 1983).

By means of reversed phase high performance liquid chromatography in the system acetonitrile - dichloromethane, the xanthophylls fatty acid esters from the purified extracts of marigold flower petals (T. erecta) were isolated on a semi preparative scale (Wolfgang Gau et al., 1983). Hatakeda et al. (1985) found a new nematicidal compound from French marigold (T. patula), which was hydroxytrementone (c -) - 2 - isopropenyl - 5 - acetyl - 6 - hydroxy- 2, 3 - dihydrobenzofuran. An alkaline water soluble nematostatic chemical constituent in the leaf extract of T. patula showed better nematostatic property at longer exposure periods against Xiphinema spp. (Indra Rajvanshi et al., 1985).

Jacobs et al. (1994) reported the synthesis and accumulation of thiophene in the parts of T. patula and T. erecta, which are responsible for the nematicidal, antiviral, antifungal and antibacterial properties of the plant. Kanagy and Kaya (1996) found that α - terthienyl at a concentration of 20 and 40 ppm significantly reduced the number of nematodes in tomato.

Bioactive extracts of the different plant parts of Tagetes exhibited nematicidal, fungicidal and insecticidal actions. The biocidal components of essential oils from the flowers and leaves are terpenoids and lutein ester contents ( Padma Vasudevan et al., 1997).

Chemical composition and antinemic activity of volatile and non-volatile fractions of T. erecta flowers were studied by Martowo and Rohana, (1987). They found that methanol extracts and essential oils of marigold flowers showed the maximum antinemic activity against M. incognita juveniles.

The ED 50 value of methanol extracts and essential oils were found as 852 and 396 μg/ml after 24h of exposure period. Maduram (1999) stated that a protein part, which inhibits the nematodes, also exists in Tagetes, thus adding one more crown to the toxicity status of Tagetes.

By keeping the above research reports in mind, a case study has been carried out to know the efficacy of Tagetes and its plant products against the root knot nematode, Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949 with the following objectives:

(i). To test the effect of different plant parts of Tagetes against hatching and mortality of root knot nematode, M. incognita in vitro.

(2). To screen the Tagetes species against root knot nematode, M. incognita under green house condition.

(3). Analysis of NPK content of Tagetes based plant products.

The experiments were conducted at the Nematology laboratory and Glasshouse of the Department of Nematology, Tamil Nadu Agricultural University, Coimbatore, India. The materials utilized and methodologies followed are described below.

Methodology

Maintenance of pure culture of Meloidogyne incognita

Required inoculum of M. incognita to raise pure culture was collected from M. incognita infested tomato roots. Roots with galls were taken and washed thoroughly with water to remove the adhering soil particles and the egg masses were separated from the roots under a Stereo Zoom microscope and transferred to a beaker filled with water for incubation and aerated intermittently. All the eggs were hatched 24h after incubation. The species identity was confirmed by following the taxonomic keys and they were used for inoculation.

Seeds of susceptible tomato cv. PKM 1 were sown in medium-sized earthen pots of 2kg capacity having steam-sterilized soil. After the germination of seeds, nematode suspension was inoculated at the rate of one J2 per gram of the soil in the rhizosphere region. The nematodes multiplied on the tomato roots were used for the further experiments.

In vitro screening of Tagetes extracts against root knot nematode, M. incognita

Tagetes varieties viz., Tagetes patula, T. erecta, T. erecta cv yellow, T. erecta cv. orange and T. minuta were screened against root knot nematode, M. incognita.

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