Abstract: Aqueous shoot extract of four weed species including Conyza canadensis, Blumea obliqua, Amaranthus viridis and Eclipta prostrata inhibited egg hatch and caused mortality of Meloidogyne javanica, the root-knot nematode juveniles in vitro to varying extent with A. viridis being the most effective. The efficacy of the powdered shoot material as soil organic amendment was tested against two nematode inoculum levels (2000 and 4000 J2 pot G 1) in a pot experiment. Soil amendment with the powdered shoot material generally reduced nematode population density, root-knot development and reproductive potential of M. javanica in brinjal roots. A. viridis was most effective in the suppression of root-knot nematode at both the nematode inoculum rates but caused slightly reduction in plant growth presumably owing to its allelopathic activity in soil.
Introduction
Soil amendments have been explored as a method of suppressing plant-parasitic nematodes. It has been shown that the efficacy of organic amendments against nematodes depends on the physical and chemical properties of the amendments (Rodriguez-Kábana, 1986). Nematicidal compounds are released by decomposing organic matter, or the compounds synthesized by the microorganism involved in the decaying process. The use of nitrogenous organic matter as soil amendment is a successful strategy for the control and management of Meloidogyne spp. and other plant-parasitic nematodes in vegetables and other root-knot susceptible crops (Mian and Rodriguez-Kábana 1982; Rodriguez-Kábana et al., 1990). Aqueous extract of various plant species reduced mobility of Xiphinima americana in vitro (Insunza et al., 2001). Shaukat and Siddiqui (2001a) found that extracts of various weeds, obtained from Karachi University Campus, caused significant mortality of M. javanica juveniles in vitro. Methanolic extract of Lantana camara produced considerable mortality of M. javanica juveniles in vitro and reduced population densities and galling intensity in mungbean roots (Ali et al., 2001). Similarly, soil amendment with Argemone mexicana substantially lowered the populations of M. javanica in the roots and rhizosphere of tomato plants under greenhouse conditions (Shaukat et al., 2002). These authors suggested that a reduced pH after soil amendment with A. mexicana was related with the inhibition of root-knot nematodes.
The objective of this study was to evaluate I) the in vitro egg hatch and nematicidal activity of the selected plant species against Meloidogyne javanica (Treub.) Chitw., ii) the influence of soil amendments with powdered shoot material of weed species including Conyza canadensis, Blumea obliqua, Amaranthus viridis and Eclipta prostrata on the development of nematode population densities in soil, root-knot infection and nematode reproductive potential and iii) the effects of such amendments on growth of brinjal (Solanum melongena L.).
Materials and Methods
Plant material and extract preparation
The Laboratory experiments were performed at the Department of Botany, University
of Karachi while greenhouse trial was conducted at the National Nematological
Research Centre, University of Karachi. The nematode antagonistic weed species
including Conyza canadensis, Blumea obliqua, Amaranthus viridis
and Eclipta prostrata were collected from the experimental field of the
Crop Diseases Research Institute, Karachi University Campus. The shoot material
of the plants was air-dried in shade and finely powdered. A 50 g powdered shoot
was soaked in 200 ml sterile distilled water and left for 72 h at room temperature.
The extract was filtered through two layers of Whatman No.1 filter paper and
kept at 6°C prior to use. To avoid bacterial contamination in the extract,
appropriate quantities of antibiotics were added.
Egg hatch activity of the plant extract
To examine the influence of plant extracts on egg hatch of M. javanica,
200±21 eggs were transferred into cavity glass slide containing 2 ml
extract of a plant species. The eggs kept in sterile distilled water served
as controls. Each treatment was replicated three times and the cavity glass
slides were randomized. After a 48 h exposure, the hatched juveniles were counted.
The eggs with intact juveniles were then transferred into cavity glass slides
containing 2 ml sterile distilled water to ascertain whether the eggs kept in
the extract had been temporarily or permanently inactivated. The juveniles that
emerged from the eggs were recounted after a further 48 h period.
Nematicidal activity of the plant extract
To assess the effects of aqueous extract of each plant species on mortality
of M. javanica, two ml of each extract was poured in a glass cavity slide
and about 38±7 freshly hatched surface sterilized juveniles of M.
javanica placed in each glass slide. Juveniles kept in sterile distilled
water served as controls. Treatments were replicated three times and dead nematodes
in each cavity slide were counted after 24 and 48 h.
Greenhouse experiments
Powdered shoot material of nematode antagonistic plants was thoroughly mixed
with sandy loam soil (72% sand, 17% silt and 11% clay; pH 8.1 and organic matter
of 0.3%) to make 25 or 50 g kg-1 (3 or 5% w/w) concentrations and
put into 8-cm-diam. plastic pots at 400 g pot-1. Soil without amendments
served as control. The pots were watered daily to promote microbial activity
so as to partially decompose the plant tissues. Following three weeks after
soil amendments, three brinjal (Solanum melongena L.) seedlings (about
8 cm tall and at two-leaf stage), raised in steam sterilized soil were planted
in each pot. The seedlings were allowed to establish for one week before soil
in each pot was inoculated by adding a total of 2000 or 4000 freshly hatched
juveniles (< one week-old) of M. javanica through four soil openings
made around each plant. Treatments and controls were replicated five times and
randomized within blocks on a greenhouse bench.
Plants were harvested at 52 days after transplant and growth parameters including plant height and fresh weight of shoot and root were recorded. Number of galls and egg masses produced on the entire root system was counted using a hand lens. Root-knot nematodes were extracted from soil (250 cc) using a modified Baermann funnel technique and counted (Rodríguez-Kábana and Pope, 1981).
Statistical analysis
Data were subjected to analysis of variance (ANOVA) and factorial analysis
of variance (FANOVA) depending upon the experimental design followed by least
significant differences (LSD) test and Duncan’s multiple range test using
STATISTICA ver. 5.0 software (Statsoft Inc. Tulsa, Oklahoma, USA, 1995). Percentage
data were transformed by an arcsine transformation prior to analysis.
Results
Egg hatch activity of the plant extract
Exposure of M. javanica eggs to shoot extract of taxonomically different
plant species resulted in a significant (p<0.05) reduction of egg hatch compared
to the controls (Table 1). When efficacy of shoot extract
of such nematode antagonistic plant species was tested, Amaranthus viridis
was found to cause maximum inhibition of egg hatch (32.46%) over the controls.
When eggs with intact juveniles were transferred from shoot extract to sterile
distilled water, hatching activity of the nematode was generally lower (except
Eclipta prostrata) in distilled water compared to the controls. Amaranthus
viridis (>32% over the controls) caused greatest permanent inactivation
of eggs followed by Conyza canadensis (>24% compared to the controls).
Egg hatch activity of the plant extract
Shoot extract of plant species caused significant (p<0.05) mortality
of M. javanica juveniles, in vitro, at both the exposure periods
(Table 2). However, the extent of nematode mortality varied
with plant species. With respect to the nematicidal activity, A. viridis
exhibited greatest nematicidal effects followed by C. canadensis at both
the time periods.
Greenhouse experiments
Soil amendments with plants caused significant (p<0.05) influence on
nematode populations (Table 3). Greatest reduction in soil
nematode population was produced by A. viridis followed by C. canadensis
and E. prostrata while B. obliqua did not reduce the nematode
population significantly.
| Table 1: | Effects of four plant species on egg hatch of Meloidogyne javanica |
| aAfter a 48 h exposure to plant extract, the egg masses were transferred to sterile distilled water | |
| Table 2: | Effects of four plant species on mortality of Meloidogyne javanica |
| Table 3: | The influence of soil amendments with powdered shoot of four plant species on nematode population densities in soil, root-knot and egg mass development due to Meloidogyne javanica on brinjal |
| Table 4: | The influence of soil amendments with powdered shoot of four plant species on growth of brinjal plants |
Generally, the nematode density in soil was observed to be greater at higher (4000 J2 pot-1) nematode inoculum. Regardless of nematode application rate, amendment with 5% shoot material resulted in greater reduction of nematode densities than did 2.5% dosage. With respect to nematode inoculum density of 2000 J2 pot-1, galling intensity was significantly lowered following soil amendment with A. viridis at both the dosages and by C. canadensis at 5% application rate only. However, at a higher inoculum density (4000 J2 pot-1), with a few exceptions, root-knot development was markedly reduced by the plant species at both the application rates. With the exception of E. prostrata at both concentrations and C. canadensis and B. obliqua at 2.5% application rate, soil amendment with shoot material of plant species significantly (p at the most 0.05) reduced egg-mass production by M. javanica on brinjal roots, at both the nematode inoculum levels.
Soil amendment with plant species did not result in any marked changes in plant height with the exception of B. obliqua where it was significantly (p<0.05) enhanced at both the inoculum levels though shoot and root weights remained uninfluenced (Table 4). In general, there was a tendency for plant height to decrease with increasing application rates of shoot material in soil. In addition, A. viridis and C. canadensis tended to reduce plant growth, particularly at higher application rate (5%) but at lower application rate (2.5%) shoot and root growth were not affected.
Discussion
Our results indicate that aqueous shoot extract of four weed species including Conyza canadensis, Blumea obliqua, Amaranthus viridis and Eclipta prostrata reduced considerable egg hatch and induced juveniles deaths of M. javanica in vitro. The weed species differed with respect to nematicidal activity suggesting that plant species posses nematicidal principle(s) that varied qualitatively and quantitatively. Since plant extracts that showed nematicidal activity, were prepared in water, the active compound(s) seem to be polar in nature. However, whether the inhibition of egg hatch and nematicidal activity found here was due to a single compound or a number of compounds can not be stated with certainty. Further investigations are needed in this regard. Similar to our study, Wang et al. (2001) observed that root leachates of Tegetes erecta caused mortality of Rotylenchulus reniformis. Husan-Bano et al. (1999) observed that methanolic extract of powdered shoot of T. patula inhibited egg hatch of M. javanica in vitro. Shaukat and Siddiqui (2001a) observed that methanolic extracts of several plant species caused significant morality of M. javanica juveniles. The observed nematicidal activity could be related with the release of phenolic and other secondary metabolites from powdered shoots of weed species. In a previous study, phenolic compounds such as caffeic acid, benzoic acid and p-coumaric acid induced juveniles deaths in M. javanica in vitro (Shaukat and Siddiqui 2001b).
In the present study, soil amendment with Amaranthus viridis reduced nematode population densities in soil, nematode reproductive potential and root-knot infection induced by M. javanica in brinjal, compared to the controls. These differences with reference to control could either be due to the changed nutrient status of the soil following amendments with plant material or because of the allelochemicals that were added to the soil either directly through the shoot material or through their products of microbial degradation. However, the observed soil suppressiveness to host plant against nematodes via changes in the fungal communities of the soil and rhizosphere can not be ruled out (Shaukat and Siddiqui, 2001c). In the current study, Blumea obliqua and Eclipta prostrata, which caused some juvenile mortality in vitro, failed to reduce nematode densities and galling in brinjal. It is plausible that B. obliqua and E. prostrata produce active nematicidal compounds that are activated only in the presence of light. Amendment of soil with organic toxicants has been used in various pathosystems by several workers under greenhouse conditions (Mankau and Minteer, 1962; Sitaramaiah and Singh, 1978; Rodríguez-Kábana, 1986; Ritzinger and MacSorley, 1998; Shaukat et al., 2002). Kheir et al. (2000) studying the effects of 18 ornamental plants, found that soil amendment with two composites, i.e., Tegetes erecta and Zinnia elegans resulted in a considerable reduction in reproductive potential and gall development by M. incognita on sunflower roots.
It is interesting to note that C. canadensis and A. viridis when applied at a rate of 5% w/w, reduced plant growth. In addition to competition for resources, some weeds interfere with the crop plants through production of chemical substances (allelochemicals) that inhibit their growth and development. The inhibitory effect of weeds may be due to a variety of allelochemicals, including phenolic acids, terpenes, terpenoids, glycosides, alkaloids and flavonoids (Blum, 1996). Alleopathic potential of A. viridis and C. canadensis has been previously reported (Narweal, 1994; Munir, 2001).
The results obtained here suggest that the application of a specific organic amendment could be exploited to keep densities of nematodes at a safe threshold level, where they may not cause any harm to the economically important crops.