2019 SCIENCELINE  
Journal of Life Science and Biomedicine  
J. Life Sci. Biomed., 9 (6): 164-169, 2019  
License: CC BY 4.0  
ISSN 2251-9939  
DOI: https://dx.doi.org/10.36380/scil.2019.jlsb26  
In vitro nematicidal activity of Juglone against  
Meloidogyne incognita race 2 infesting pomegranate  
Dama Laxmikant  
Department of Zoology, D.B.F. Dayanand College of Arts and Science, Solapur- 413002 (M.S.), India  
Corresponding author’s Email: southraj@gmail.com; ORCID: 0000-0003-3157-8457  
ABSTRACT  
Original Article  
Introduction. Juglans regia L. (walnut) is also known as the medicinal plant and native to the  
mountain ranges of central Asia. Recently, a severe infestation of root-knot nematodes  
(RKN, identified as Meloidogyne incognita race 2), was observed on Punica granatum L.  
(pomegranate). Aim. The present study aimed to investigate the effect of in-vitro nematicidal  
activity of the purified bioactive compounds (Juglone: 5-Hydroxy 1, 4-Napthoquinone)  
isolated from medicinal plant like walnut on Meloidogyne species associated with  
pomegranate under field conditions. Methods. Plants are cultivated at the agricultural farm  
PII: S225199391900026-9  
Rec. 10 October 2019  
Rev. 15 November 2019  
Pub. 25 November 2019  
Keywords  
in Solapur, (M.S.), India. Results. Roots of infected plants were heavily galled and soil 5-Hydroxy 1, 4-  
samples collected from the affected plants second stage juveniles (J2). The ability of Napthoquinone,  
treatment schedule of Juglone was tested using an in-vitro method against RKN - Nematicidal activity,  
Meloidogyne incognita race 2 infesting pomegranate. Juveniles in the control (distilled water; Root-knot nematode,  
carbofuran) were compared with treated groups. The Juglone showed a 100% mortality in 5 Punica granatum L.  
µl/ 10 ml of distilled water/ 100 nematodes.  
INTRODUCTION  
Pomegranate (Punica granatum L.) is an important fruit crop of India. Pomegranate is delicious, popular fruit in  
India and several other countries of the world. It is the natural food for man. It is excellent source of vitamins,  
minerals, and enzymes. All parts of the fruit, seed, rind, and membrane have been shown to have potential  
health benefits. One of the most studied constituent of pomegranates is a polyphenol calls ellagitannins and  
ellagic acid [1].  
Nematodes associated with pomegranate trees causes’ severe damage to whole thickness of the root and  
stunting. Lesions were formed at the site of entry with necrosis, enlargement of cells into giant cells, containing  
egg masses and sections of larvae [2].  
There are different pest attacks on the pomegranate plants by bacteria, virus fungal, but plant parasitic  
nematode is one of the major yield limiting factors. Now the productivity of pomegranate in Maharashtra is  
highest in the country [3, 4]. Further, Meloidogyne incognita L. has been observed as a serious problem on  
pomegranate in Maharashtra and attributed 32% yield losses in the identified hotspots. In the present study, in  
vitro nematicidal activity of Juglone as bioactive compound was evaluated on root-knot nematodes collected  
from pomegranate. Juglone, also called 5-hydroxy-1,4-naphthalenedione (IUPAC) or 5-hydroxynaphthoquinone,  
is an organic compound with the molecular formula C10H6O3. In the food industry, Juglone is also known as C.I.  
Natural Brown 7 and C.I. 75500; with systematic name: 5-Hydroxy-1,4-napthoquinone.  
MATERIAL AND METHODS  
Preparation of plant extracts  
Juglone, extracted by using polar and non-polar chemicals by cold extraction method; extract will be  
separated by using Column chromatography and tested by spectroscopy as follows: IR = Perkin-Elmer 599-B in  
CHCl3; NMR = 200 MHz Brucker Ac 200 MHz in CDCl3; Mass Spectra, Finnigan Mat 1020 usind direct inlet  
system at 70 V; For purity and structure determination of the pure molecules Juglone = 5-Hydroxy 1, 4-  
Napthoquinone.  
Citation: Laxmikant D. In vitro nematicidal activity of Juglone against Meloidogyne incognita race 2 infesting pomegranate. J. Life Sci. Biomed., 2019; 9(6): 164-169;  
www.jlsb.science-line.com  
164  
Collection of root samples  
Pomegranate (P. granatum L.) root samples with soil were collected from the field located in Solapur  
district, Maharashtra state, India. Two hundred grams of soil and roots from each sample were processed for  
the isolation of nematodes.  
Cobb’s decanting and sieving method for collection of nematodes  
Nematodes were extracted by Cobb’s decanting and sieving methods [4]. Direct contact toxicity of  
compound at different dose was analyzed by exposing freshly hatched J2 and J3 of Meloidogyne species [5].  
In vitro phyto-nematicidal activity  
For in vitro nematocidal activity, the method described by Dama [6] and Dama et al. [7]. used for present  
study. The test animals are divided in 5 groups in which one distilled water and one for slandered carbofuran  
chemical, each group contains 100 phyto-nematodes, with test compound concentration of 1 µl to 5µl.  
The observations were taken at 24 hrs after exposure of test compounds under compound and  
stereomicroscope, on Juvenile mortality was recorded and expressed as percentage of efficacy against  
Meloidogyne species in each group were estimated by statistical analysis on the basis of the arithmetic mean  
number of J2 recovered. Juveniles in the control (distilled water; carbofuran), and also treated groups were  
compared statistically.  
Statistical analysis  
For taxonomic identification, the generated data was compared with taxonomic aids. Rate of immobility  
(i) was calculated and the obtained data analyzed by using Standard statistical methods (like SAS method).  
Standard deviation or confidence intervals of the means of LC50 values are calculated and recorded. Formula  
for percentage of mortality is as follows:  
Percentage of mortality =  
Number of dead larvae  
× 100  
Number of larvae introduced  
RESULTS AND DISCUSSION  
The Results are shown in table 1 and figures 1-6. The action of Juglone on Meloidogyne species associated with  
Pomegranate roots, demonstrated maximal activity. The spectral data of Juglone where shown in figure 1.  
The spectral data analysis of Juglone are as follows: IR= CHCl3 : 3300, 1730, 1430 cm-1; NMR= 1H mr (200  
MHz); CDCl3 : 6: 92 s(2H); 7.30m (1H) and 7.72m (2H). 11.9 (s, 1H, OH, D2O exchange); Mass Spectra 174.  
The results of spectral data namely IR, NMR and Mass Spectra showed the isolated pure bioactive  
compound as Juglone. Figure 2 shows the Juglans regia tree with green walnuts. Punica granatum L., the healthy  
plant, infected plant and infected roots were shown in figure 3. Figure 4 showed the extraction of plant  
paparasitic nematode by using Cobb’s decanting and sieving method for collection of Nematode.  
Phyto-nematicidal activity of Juglone on the mortality (immobilized nematodes) of root-knot nematode  
Meloidogyne sp. in 24 hrs were shown in table 1 and figure 6. The results compared with the available  
observations pointed out by Shelke and Darekar [8] and Pawar et al. [3]. Although, observations suggested that  
Juglone was active compound which require future pharmacological and toxicological studies prior to their use  
in the integrated diseases management (IDM) of plant parasitic nematodes. The result indicated that Juglone  
was very effective to controlling Meloidogyne species 100% mortality in 5 µl.  
The present work confirms that the effect of Juglone shows the phyto-nematicidal activity on M. incognita  
race II associated with Pomegranate at very low concentration. The uses of bioactive compounds are an  
interesting option to control the root-knot nematodes instead of the chemical control and have no effect on  
human health. There was no any reported information on the effect of Juglone on plant‐parasitic nematode M.  
incognita race 2 infecting pomegranate. The seasonal incidence and biological management of root-knot  
nematode, Meloidogyne incognita [9], the infesting pomegranate, Punica granatum observed by Walunj [10].  
Citation: Laxmikant D. In vitro nematicidal activity of Juglone against Meloidogyne incognita race 2 infesting pomegranate. J. Life Sci. Biomed., 2019; 9(6): 164-169;  
www.jlsb.science-line.com  
165  
a) IR  
b) NMR  
c) Mass Spectra  
Figure 1. Spectra of Juglone. a) IR; b) NMR; c) Mass Spectra  
Figure 2. Juglans regia tree shows green walnuts  
Citation: Laxmikant D. In vitro nematicidal activity of Juglone against Meloidogyne incognita race 2 infesting pomegranate. J. Life Sci. Biomed., 2019; 9(6): 164-169;  
www.jlsb.science-line.com  
166  
a
b
c
Figure 3. Punica granatum L.; a) healthy plant, b) infected plant, c) infected roots (root-knots of Meloidogyne spp.)  
Figure 4. Cobb’s decanting and sieving method for collection of nematodes.  
a
b
c
Figure 5. Meloidogyne sp. isolated from the soil and roots from Punica granatum L., (Root-knot nematodes- M.  
incognita race 2, magnification 10x × 45x); a) Juveniles, b) Female, c) Nematode entering in the pomegranate root  
Citation: Laxmikant D. In vitro nematicidal activity of Juglone against Meloidogyne incognita race 2 infesting pomegranate. J. Life Sci. Biomed., 2019; 9(6): 164-169;  
www.jlsb.science-line.com  
167  
Table 1. Phytonematicidal activity of Juglone on the mortality (Immobilized Nematodes) of root-knot nematode  
Meloidogyne sp. in 24 hrs  
Concentration of Juglone / 10 mal of D.W.  
1 µl  
2 µl  
3 µl  
4 µl  
5 µl  
No. of root-knot Nematodes  
Juglone  
Distilled water  
Carbofuran  
100  
60  
00  
80  
100  
74  
00  
85  
100  
84  
00  
96  
100  
92  
00  
100  
100  
00  
100  
100  
120  
No. of root-knot Nematodes  
Juglone  
Distilled water  
Carbofuran  
100  
80  
60  
40  
20  
0
1 µl  
2 µl  
3 µl  
4 µl  
5 µl  
Concentration of Juglone/ 10 mal of D.W.  
Figure 6. Phyto-nematicidal activity of Juglone on the mortality (immobilized nematodes) of RKN nematode  
Meloidogyne spp. in 24 hrs.  
CONCLUSION  
Juglone is highly active phyto-nematicide as compared to control and other phyto-nematicide. Improved data  
management and decision-support systems should facilitate the integration of new and traditional IPM  
strategies and tactics. In conclusion the napthoquninones are highly active against in controlling plant parasitic  
nematodes namely Meloidogyne Sp. To reduce the cost for collecting the napthoquinones from various plant  
other raw materials like, Tendu leaves, Plumbago zylanica and leaves of Lawsonisa innermis need to be further  
carried out. This research work findings are useful for plant pathologists, teachers, agriculturist, students,  
farmers and research workers in this field of study.  
DECLARATIONS  
Acknowledgements  
We thank to Principal, Dayanand College of Arts and Science, Solapur for providing laboratory facilities.  
The authors gratefully acknowledge University Grants Commission (UGC) New Delhi (India) for financial  
assistance provided to carry out this work through major research project (F. No- 39-645/2010 (SR) Dated 10-01-  
2011).  
Authors’ Contributions  
All authors contributed equally to this work.  
Competing interests  
The authors declare that they have no competing interests.  
Citation: Laxmikant D. In vitro nematicidal activity of Juglone against Meloidogyne incognita race 2 infesting pomegranate. J. Life Sci. Biomed., 2019; 9(6): 164-169;  
www.jlsb.science-line.com  
168  
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Citation: Laxmikant D. In vitro nematicidal activity of Juglone against Meloidogyne incognita race 2 infesting pomegranate. J. Life Sci. Biomed., 2019; 9(6): 164-169;  
www.jlsb.science-line.com  
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