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Nematicidal Effects of Acacia nilotica, Azadirachta indica, Brassica chinensis and Ecklonia maxima against Soybean Cysts Nematode.

Byline: M. Auwal Hassan, Shi Hongli, Nazim Hussain, Hoa Pham Thi and Zheng Jingwu

Abstract

The effect produced by aqueous extract of Acacia nilotica, Azadirachta indica, Brassica chinensis, and Ecklonia maxima in the control of soybean cysts nematode (SCN) was tested in laboratory and green house. SCN eggs, second stage juvenile and soybean plants inoculated with 1000 eggs were treated with extract of each of the plant material at concentrations of 120 ug/mL and 12 ug/mL. Results obtained showed all plant materials significantly reduced H. glycine population compared to control. A. indica at concentration of 120 ug/mL exhibited highest inhibition to egg hatch, juvenile mortality, reduction in number of females and cysts of 75.21%, 100%, 83.12% and 80.17%, respectively. This was closely followed by B. chinensis, A. nilotica and E. maxima.

The aim is to study the efficacy of each plant material for the possibility of mixing their crude form to formulate an organic nematicide alternative to methyl-bromide that was complete banned globally in 2005 due to its harmful effect on the ozone layer. A. indica and B. chinensis showed high potential nematicidal properties, which could be explored further in effort to formulate organic nematicide from mixture of two the two plant materials in order to benefit from synergistic and potentiating effect. (c) 2013 Friends Science Publishers

Keywords: Heterodera glycines; Glycine max; Alternative control

Introduction

Currently, the cyst nematodes (Heterodera spp.) are considered most important soil borne pathogens of crops throughout the world (Wrather and Mitchum, 2010; Hajihasani et al., 2011). Soybean cysts nematode (Heterodera glycines Ichinohe, 1952) is considered a devastating pest in soybean growing areas of the world (Wrather and Mitchum, 2010). Being a cyst producing nematode, it has a high survival and dissemination capacity, making its control difficult. Chemical nematicides are the main control method used by farmers. Nematicides although efficient and drastic in action are now being reappraised due to hazards they pose to the environment. De-registration of some of the more hazardous nematicides, for instance, the world wide phasing out of methyl bromide completed in 2005 due to its effect on the atmospheric ozone layer has removed the charm many growers relied upon for over forty years for control of plant parasitic nematodes (Sikora, 2002).

Discovery of plant materials based nematicidal substances lend a boost to safe and sustainable alternative approaches for nematodes control (Chitwood, 2002; Rehman et al., 2006; Safdar et al., 2012). Numerous biocidal "active principles" extracted from plants have exhibited efficacy against different types of pathogens including plant parasitic nematodes (Bones and Rossiter, 2006; Kabeh and Jalingo, 2007; Elbadri et al., 2009; Khan et al., 2009; Sultana et al., 2011). However, there is comparatively little information on use of plant materials to control cyst forming nematodes on field crops such as cereal and pulses.

Therefore, the objective of this study is to assess the effect of applying cold aqueous extracts of acacia (Acacia nilotica), Chinese cabbage (Brassica chinensis), neem (Azadirachta indica), and sea bamboo (Ecklonia maxima) in suppressing population of soybean cyst nematode (Heterodera glycines) and to evaluate the concentration of materials needed to effectively cause reduction un population of H. glycines without any phyto-toxicity effects on the crop.

Materials and Methods

Milled A. indica kernel, A. nilotica resin, B. chinensis leaves and E. maxima were bought from China Ocean University Organic Fertilizer Manufacturing Company, Qingdao, China. Each plant material was weighed, 30 grams, wrapped in a 1 mm diameter mesh cloth and immersed in 200 mls of sterilized distilled water (SDW) and allow to stay for 24 hours (Kumar, 2003). The water extract was obtained by filtering the mixture (plant material and SDW) through a Whatman # 42 filter paper (Iqbal et al., 2001). Filtrates were made up to 250 mls using SDW to give a concentrations of 0.12 mg/L, which were subsequently referred to as standard concentrations (SC) and were serially diluted with SDW to give two concentrations of 120 ug/mL and 12 ug/mL used in experiments. Soybean variety highly susceptible to H. glycines ("Mudanjiang GB1352") was planted in H. glycines infested soil to culture the inoculums. Whenever the need arise, soybean which was uprooted and matured cyst collected from the plant roots and soil.

The H. glycines cysts were extracted from the soil sample using a modified fenwick can (MFC) flotation method (Fenwick, 1940). To prepare the test plant, soybean seeds are placed on a large petri-dish covered with a lid and placed inside a growth chamber (25-27oC), germinated seeds were used in pot experiments.

Laboratory experiments were conducted to study the mechanism of nematodes suppression by the four plant aqueous extracts. Aliquots of 4 mls filtrates of each of the plant material at concentrations of 120 ug/mL and 12 ug/mL and 1 mL of SDW containing 200 eggs/mL of H. glycines suspension was loaded into Bureau of Plant Industry (BPI). Each BPI dish was placed inside a petri-dish (6 cm diameter) and covered with lid. SDW was used in place of extracts for control. To inhibit bacterial attack on the eggs, 0.3 mL of 1.0% streptomycin sulfate was added to each treatment (Hassan et al., 1981). Petri-dishes containing treatments were arranged in complete randomized design (CRD) inside a growth chamber at temperature of 25-27oC. Hatched second stage juveniles (J2) of H. glycines were counted under a light microscope at intervals of 48, 60, 72, 84 and 96 h. The experiment was replicated four times and repeated twice. Percentage H. glycines egg, hatch inhibition was determined using formula shown below:

1 - (N in T after treatment) Percentage egg inhibition (%) = ------------------------------------------ x 100

N in Control after treatment

Where: N = Number of eggs; T = Treated.

A second experiment was conducted to test the effect of the extracts on H. glycines juveniles. Aliquots of 4 mls of each plant material at concentrations of 120 ug/mL and 12 ug/mL and 1 mls SDW of H. glycines juveniles suspension containing 20 J2s were loaded into a BPI dishes, these were incubated inside a growth chamber at 26 - 28oC. Treatments were observed under a light microscope and a picking hair was used to slightly touch the J2, those that did not respond to the touching sensation were counted as dead at time intervals of 48, 60, 72 and 84 h. Sterilized distilled water was used to replace extract as control. The experiment was replicated four times and repeated twice. Percentage J2 mortality was calculated using the formula shown below:

(Mortality % in Treated - Mortality % in Control) Percentage J2 mortality (%) = -------------------------------------------------------------- x 100

100 - Mortality % in Control

In green house soybean seedlings were transplanted at the rate of one seed per pot (20 cm in diameter, 15 cm deep) containing a mixture of sterilized sandy soil (50 % sand, 30% loam and 20% clay). Each plant was inoculated with 1000 eggs of H. glycines. A period of 1 week is allowed to elapse after inoculation and 100 mls of extracts were used to drench the soil in place of tap water supply. Distilled water only served as control. Eight weeks after planting, soybeans were up rooted, roots were excised from shoots and washed in a gently flow of tap water. Using a stereomicroscope the number of H. glycines females and cysts were counted per one gram root per plant. Data was taken shoot height, root length, dry shoot weight and dry root length. The experiment was replicated four times and repeated twice.

Data Analysis

Data were analyzed using a one- way analysis of variance (ANOVA) conducted using Michigan State University statistical software (MSTAT-C, 2000). Significant differences among the treatment means was separated using Fisher's protected least significant difference (LSD) at 95% level of confidence.

Results

Aqueous extracts of all four plant materials at concentrations of 120 ug/mL hindered significantly (P<0.05) H. glycines egg hatch compared to control. Percentage egg hatch inhibition after interval of 96 hours for A. indica, B. chinensis, A. nilotica and E. maxima was 75.21%, 62.90%, 41.80% and 26.23%, respectively (Fig. 1a). Similar trend, although at lower percentage inhibition was observed at low concentration of 12 ug/mL. SDW control treatment did not hinder egg hatch.

All plant material extracts significantly (P < 0.05) differ from, and caused higher percentage mortality of J2 than the control. Aqueous extract of A. indica at concentration of 120 ug/mL at an interval of 84 h caused 100% percentage mortality of J2, this is followed by B. chinensis, A. nilotica and E .maxima that caused J2 mortality of 68.34%, 43.34% and 23.34%, respectively (Fig. 1b). Similar trend was observed at lower concentration of 12 ug/mL.

Effect of Extracts on H. glycines and Soybean

Application of all four plant material aqueous extracts at concentrations of 120 ug/mL and 12 ug/mL significantly (P<0.05) hindered H. glycines development on soybean roots compared to control. At concentration of 120 ug/mL, percentage H. glycines female development hindrance of A. indica, B. chinensis, A. nilotica and E. maxima were 83.12, 66.23, 50.65 and 40.26%, respectively compared to control treatment that exhibit 0% hindrance to female development. Similar trend was observed at the low concentration of 12 ug/mL (Fig. 2).

Soybean growth parameters of shoot height, root length, dry shoot and root weights at treatment concentration of 120 ug/mL of all four plant materials were significantly (P < 0.05) increased compared to control treatment. However, A. indica exhibited significantly (P<0.05) highest mean shoot height of 68.7 cm, followed by E. maxima, B. chinensis and A. nilotica, which gave mean shoot heights of 61.2 cm, 59.7 cm and 43.2 cm compared to control treatment, which gave significantly (P < 0.05) the lowest mean shoot height of 32.3 cm. Other growth parameters of mean root length, dry shoot and root weight more or less follow a similar trend as showed in Table 1.

Discussion

This study has demonstrated all four plant aqueous extracts

Table 1: Effect of plant materials on growth of soybean inoculated with 1000 eggs of H. glycines

Plant material###Mean Shoot###Mean Root###Dry shoot###Dry root

Conc. (ug/mL)###Height (cm)###Length (cm)###Weight (g)###Weight (g)

An 120###43.22d###10.50bc###0.60d###0.33d

An 12###36.73e###8.98d###0.48e###0.26e

Bc 120###59.68b###11.43b###1.25ab###0.45c

Bc 12###54.33c###9.63c###0.85c###0.35d

Ai 120###68.70a###13.80a###1.40a###0.65a

Ai 12###58.18b###9.95c###0.93c###0.47c

Em 120###61.25ab###11.30b###1.08b###0.53b

Em 12###54.58c###9.83c###0.83c###0.33d

Control###32.30e###7.87e###0.40e###0.18e

Each value is a mean of two trials with four replicates

Means followed by the same letter within each column are not significantly different (P < 0.05) as indicated by Fisher's least significant difference (LSD) test An= Acacia nilotica; Bc = Brassica chinensis; Ai= Azadirachta indica; Em= Ecklonia maxima Control = Sterilized distilled water (SDW) of plant materials significantly (P < 0.05) more effective in inhibiting egg hatch, causing J2 mortality and reducing number of cysts and females of H. glycines compared to control. Nevertheless, A. indica and B. chinensis exhibited significantly (P<0.05) more nematicidal properties against H. glycines population compared to A. nilotica and E. maxima. The pot experiments showed all four plant material extracts to a different extent reduced H. glycines cysts and female numbers; this apparently caused an increase in growth parameters of the soybean.

The study revealed that type of plant from which the extract was produced influenced to large extends the activity of the plant aqueous extracts against the H. glycines population, a similar observation made on M. incognita J2 (Rotim and Moens, 2005). The study also indicated that different concentration of the plant aqueous extracts exhibited different nematicidal efficacy.

This nematicidal activity is related to presences of several triterpenoids present in the plant seeds such as azadirachtin A, Azadirachtin B, Azadirachtin H, desacetylnimbin, nimbin, salannin. Plants in the genus Brassica spp. all contain glucosinolates (Fahey et al., 2001), which are hydrolyzed by specific endogenous thioglucosidases to yield a variety of biologically active products, including nitriles, thiocyanates, and isothiocyanates - ITCs (Bones and Rossiter, 2006). ITCs are related to compounds found in chemical soil fumigants metham sodium and dazomet, which release methyl isothiocyanate into the soil; this could be a valuable component of a methyl bromide alternatives program (Fahey et al., 2001). In the laboratory experiments, extracts of A. nilotica at concentration of 120 ug/mL showed promising nematicidal effect and ranked third after A. indica and B. chinensis.

This is in consonance with a study conducted by Sultana et al. (2011) on the control of Meloidogyne spp. using A. nilotica whereby 100% J2 mortality was obtained after 48 h exposure. In the pot experiments A. nilotica reduced the number of H. glycines cysts and females by half at high concentration of 120 ug/mL; this is in conformity with a green house pot study conducted by Elbadri et al. (2009) on control of M. incognita on water melon Citrullus lanatus Thunb.). Sea bamboo is the least in nematicidal properties. Paradoxically, it caused high increase in growth parameters of soybean second only to neem. This contradictory situation can be attributed to the fact that sea bamboo add nutrients to the soybean compared to extracts of the other plant materials. This makes the soybean more robust and tolerant to the H. glycines attack. This fact as elucidated by Oka (2010) showed addition of organic amendments somehow makes plants more tolerant or resistance to parasitism by nematodes.

Sea bamboo has been implied to induce resistance to abiotic and biotic stresses including nematodes parasitism (Khan et al., 2009). In conclusion, A. indica and B. chinensis have immense potential to contribute to the integrated management of H. glycines population on soybean. Further research is essential in order to quantify the efficacy of the plant materials under field conditions to enhance its utilization. This can be explored further in effort to formulate organic nematicide from a mixture of two or more plant material in order to benefit from synergistic and potentiating effect of a mixture of different materials.

Acknowledgements

The research was supported by Special Fund for Agro-scientific Research in the Public Interest in China (No, 200903040 and 201103018), and the authors wish to express their profound gratitude to Chinese government (CSC No. 2008566013) for conduct this study.

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(Received 17 September 2012; Accepted 04 December 2012)
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Author:Hassan, M. Auwal; Hongli, Shi; Hussain, Nazim; Thi, Hoa Pham; Jingwu, Zheng
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9CHIN
Date:Jun 30, 2013
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