EVALUATION OF PLANT EXTRACTS FROM NORTHEAST LIBYA FOR THEIR NEMATICIDAL ACTIVITY AGAINST THE DAGGER NEMATODE, Xiphinema index

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_________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

KEYWORDS

Biological control

Nematicidal activity

Extraction

Phonecian juniper

Mastic tree

Strawberry tree

Phillyrea

Carob

Xiphinema index

ABSTRACT

Xiphinema index is one of the most important plant-parasitic nematode that is especially damaging the grape and fig orchards by its direct feeding or from the viruses that it transmits. The nematicidal activities of fifteen plant extracts were tested in vitro against adult females of this nematode. The extracts were isolated from leaves and small branches of five endemic trees viz Phoenician juniper (Juniperus phoenicea L.); mastic tree (Pistacia lentiscus L.); strawberry tree (Arbutus pavarii Pamp.); Phillyrea (Phyllarea angustifoliaL.) and Carob (Ceratonia siliqua L.)] from the Al-Jebel Akhdar region of northeast Libya. Three solvents i.e. water, ethanol, and hexane were used to isolate the extracts. Highest nematicidal activity was reported from the ethanolic extract of J. phoenicea (27 minutes for LT50 and 47 minutes for LT90), followed by hexane extract of Pistacia lentiscus L. with 232 and 350 minutes for LT50 and LT90, respectively. The activities of other extracts were significantly less than these two extracts.

Mohamed AM Adam* and Abdelkrim Amer

Plant Protection Department, Agriculture Faculty, Omar Al-Mukhtar University, P. O. Box 919 Al-Beida, Libya.

Received – August 01, 2014; Revision – August 17, 2014, Accepted – October 03, 2014 Available Online – October 25, 2014.

EVALUATION OF PLANT EXTRACTS FROM NORTHEAST LIBYA FOR THEIR

NEMATICIDAL ACTIVITY AGAINST THE DAGGER NEMATODE,

Xiphinema index

E-mail: m_a_m_adam@yahoo.com (Mohamed AM Adam)

Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

* Corresponding author

Journal of Experimental Biology and Agricultural Sciences, October - 2014; Volume – 2(5)

Journal of Experimental Biology and Agricultural Sciences

http://www.jebas.org

ISSN No. 2320 – 8694

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1 Introduction

Plant-parasitic nematodes are one of most important plant pathogenic agents. More than 4,100 described species to date (Decraemer & Hunt, 2006) represent an important constraint on global food security. Yield loss caused by plant nematodes has been estimated at 157 billion US$ per year (Abad et al., 2008). The family Longidoridae includes some 480 species and the subfamily of Xiphinematinae with one genus Xiphinema comprises 258 species (Oliveira & Neilson, 2006). One of these species, Xiphinema index (Thorne & Allen, 1959) is an important parasite of grape and due to its worldwide distribution it’s also works as an important vector of the fan-leaf virus which causes a serious disease of the grape. X. index specifically transmits grapevine fan leaf virus (GFLV) from infected plants to healthy grape.

X. index is thought to have been dispersed from the Middle East into other grapevine-growing regions of the world in soil accompanying propagation material where it has become an invasive agricultural, soil-borne pathogen (Brown & Taylor, 1987). This species has been reported in several countries, most recently in Libya also (Adam & Saleh, 2010). Nematode control is usually based on chemical nematicides which present potential risks to farmers, consumers, non-target organisms and the environment; therefore, many researchers search for natural products with nematicidal activity such as, root exudates or plant volatile compounds (Linford et al., 1938; Dechet,1991; Sasnelli & Catalano1991;Insunza et al., 2001a). In present present study fifteen plant extracts using three solvents(water, ethanol and hexane) from five endemic plant species were first time in vitro screened for their nematicidal activity against adult females of X. index.

2 Materials and Methods

2.1 Preparation of Plant extracts

Fifteen plant extracts were prepared from five plants and three different solvents viz distilled water, ethanol (99.9%) and hexane (99.9%) (Table 1). These are endemic in the Al-Jebel Akhdar region northeast Libya. Plant materials (leaves and small branches) were picked from healthy trees around the study area. They were washed with distilled water and allowed to dry for 48 h at room temperature before extraction.

2.1.1 Water extracts

100 grams of each sample were ground with 200 ml distilled water in a blender (16,000 rpm.) for ten minutes at a ratio of 1:2 (w/v). The crushed samples were filtered under vacuum through filter paper (Whatman no. 40; Whatman International Ltd., Maidstone, UK), placed on a glass plate 9 cm diameter and stored in a drying chamber for 24 hrs.

2.1.2 Ethanol extracts

200 ml of 99.9% ethanol were added to the plant materials that remained on the filter paper after the preparation of water extract. The ethanol extract was placed on a glass plate 9cm in diameter and stored in drying chamber for 6 hrs.

2.1.3 Hexane extracts

200 ml of hexane were mixed with the plant materials that remained on the filter paper after the water extraction. The hexane extract was stored in a drying chamber for 6 hrs in glass plate with a diameter of 9 cm.

All of the dried extracts were considered as 100% concentration extracts and were re-dissolved in distilled water at a 25% concentration for use in bioassays.

2.2 Nematode extraction

Individuals of X. index were extracted from soil samples collected from vineyards of the Algariga region (near Al-Beida city). Collected samples were processed by Cobb’s sieving and decanting method (Southey, 1986). The nematodes were caught in the 250μm sieve and 10 adult females were hand picked with a small needle and placed in 5 cm diameter Petri dish.

2.3 Extracts bioassay

Bioassays were conducted in 5cm petri dishes containing 10 adult females of X. index and 5 ml of 25% concentration of the tested extract (3.75 ml distilled water and 1.25 ml volume of the test solution) and kept in the dark at 20 ± 1°C (Argentieri et al., 2008). After different times, the nematodes were observed with a dissecting microscope. Nematodes were considered immobile if they failed to respond to stimulation with a needle.

Table 1The selected flora for the preparation of various extracts used in study

Scientific Name

Local Name

Local Name in English

Common Name SN

Juniperus phoeniceaL.

رعرعلا AL-ArAr

Phoenician juniper

1

Pistacia lentiscus L. موطبلا

AL-Batom Mastic tree

2

Arbutus pavarii Pamp.

ير مشلا AL- Shmary

Strawberry tree

3

Phyllarea angustifoliaL.

خسلا AL-Sakab

Phillyrea

4

Ceratonia siliqua L.

بورخلا AL-Carob

Carob

5

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Figure 1 The Lt50 and Lt90 value with error bars of five water extracts against adult females of Xiphinema index Thorne and Allen, 1959.[1 = Phonecian juniper (Juniperus phoenicea L.), 2 = mastic tree (Pistacia lentiscus L.), 3 = strawberry tree (Arbustus pavarii

Pamp.), 4 = Phyllarea (Phyllarea angustifolia L.), 5 = carob (Ceratonia siliqua L.)]

Figure 2 The Lt50 and Lt90 value with error bars of five ethanol extracts against adult females of Xiphinema index Thorne and Allen, 1959. [1 = Phonecian juniper (Juniperus phoenicea L.), 2 = mastic tree (Pistacia lentiscus L.), 3 = strawberry tree (Arbustus pavarii

Pamp.), 4 = Phyllarea (Phyllarea angustifolia L.), 5 = carob (Ceratonia siliqua L.)]

Immobile nematodes were re-suspended for 24 hrs in distilled water, In the case that immobile nematodes regained mobility after this time the effect was considered as nematostatic, The treatment were replicated three times and three untreated controls using pure distilled water for water extracts, distilled

water with 0.0005% ethanol for ethanol extracts, and distilled water with 0.0005% hexane for hexane extracts. The Lt50 and

Lt90 of each extracts were calculated through account of dead

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The calculation of Lt50, Lt90 and statistical analysis of these

values (the exposure time at which 50% and 90%, respectively, of the adults were died) were calculated by probit analysis using the PROBIT software Statistical Package for the Social Sciences. The overlapping 90% of error bars of confidential limits were used to evaluate the degree of significant differences between treatments (Scoot et al., 1986; Cochran, 1989; Jensen,1993)

3 Results and discussion

The resulting Lt50 and Lt90 values, calculated from exposure time of nematodes to defined dose and concentration from each extracts are illustrated in Figures 1-3. Whereas, No death was recorded in the controls until 72 hrs.

3.1 Water Extracts

Extract from A. pavarii (Figure1) had the highest nematicideal activity in the water extracts with an Lt50 value of 350 minutes and an Lt90 at 570 minutes; whereas the least active extract was from C. siliqua (Figure 1) with an Lt50 of 1,970 minutes and Lt90 of 3,610 minutes.

3.2 Ethanol extracts

These extracts had good nematicidal activity especially in J. phoenicea with the shortest Lt50 of 27 minutes and 47 minutes

for Lt90; whereas, the longest Lt50 and Lt90 were recorded for the extract from C. siliqua viz 860 and 1,460 minutes respectively (Figure.2).

3.3 Hexane extracts

The nematicidal activity of extracts of hexane was between that of water and ethanol; where the highest Lt50 was is 232 minutes and 350 minutes for Lt90 was registered P. lentiscus and the lowest was reported from C.siliqua i.e. 1,900 minutes and 3,500 minutes for Lt50 and Lt90 values respectively (Figure 3).

The ethanol extract from J. phoenicea had the highest activity against the adult females of X. index through 27 minutes for Lt50 and 47 minutes for Lt90 followed the hexane extract from P. lentiscus with 232 minutes and 350 minutes for Lt50 and Lt90 respectively.

These plant extracts were used in vitro for the first time against plant parasitic nematode. The results showed significant differences in Lt50 and Lt90 between the plant species and the kind of solvent (Table 2). This result was agreed with the most of other researches such as the study conducted by Insunza et al. ( 2001b) who found that 18 out of 21 plant species extracts effect on Xiphinema americanum sensu lato in vitro.

Figure 3 The Lt50 and Lt90 value with error bars of five hexane extracts against adult females of Xiphinema index Thorne and Allen, 1959. [1 = Phonecian juniper (Juniperus phoenicea L.), 2 = mastic tree (Pistacia lentiscus L.), 3 = strawberry tree (Arbus

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Table 2Comparisons of nematicidal activity of extracts using three solvents from five plants on X. index .

Lt90

Lt50

Solvents No of extracted plants as in Tab. 1

610 a

320a

Hexane

1

47 a

27 a

Ethanol

1400 ab

720ab

Water

350 a

232 a

Hexane

2

1100 a

600 ab

Ethanol

1390 ab

850 ab

Water

693 ab

410 ab

Hexane

3

575 a

320 a

Ethanol

570 a

320 ab

Water

950ab

540 ab

Hexane

4

640 a

375 a

Ethanol

630 a

370 ab

Water

3500 c

1900c

Hexane

5

1460 b

860b

Ethanol

3610 c

1970 c

Water

No significant differences between the treatments with the same letter for Lt50 and Lt90 separately.

Moreover the study done by Wiratno et al., (2009) on evaluated of nematicidal activity of 17 plant extracts against Meloidogyne incognita found that, tobacco, clover betelvine and sweet flag extracts are the most effective in killing the nematode. Similarly, Abbas et al., (2009) reported that, ethanol extracts was found better than aqueous extract in mortality of second stage juveniles of Meloidogyne javanica. The Xiphinema index is ectoparasitic migratory nematode whereas Meloidogyne spp. are endoparasitic sedentary nematodes that secret protect proteins against plant defense to protect themselves. In present study the extract of J. phoenicea may be targeted for advanced studies to evaluate its effectiveness on a large scale in the field.

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