Research Article - (2022) Volume 9, Issue 3
Received: 06-Sep-2022, Manuscript No. IJNEOAJ-22-73918; Editor assigned: 09-Sep-2022, Pre QC No. IJNEOAJ-22-73918; Reviewed: 23-Sep-2022, QC No. IJNEOAJ-22-73918; Revised: 07-Nov-2022, Manuscript No. IJNEOAJ-22-73918; Published: 14-Nov-2022
Nematicidal activity of an aqueous extract from dried leaves of Eucalyptus globulus was evaluated towards the root lesion nematode Pratylenchus vulnus in vitro and pot experiment. In both experiments, the application of four concentrations of the leaf aqueous extract (100%, 60%, 30%, 15% w/v) significantly reduced the number of females and males on apple rootstock MM106 roots. In vitro test showed that the highest net mortality was recorded with the concentration 100% (96%) after 72 hrs of exposure time. Pot experiment studies indicated that the concentration 100 increased significantly (P <0.05) the female and male reduction rates (84.43% and 91.40% respectively) compared to others leaf extracts concentrations. The chemical treatment with Oxamyl G (Oxamyl granule) reduced significantly female and male rates by 98.30% and 100%, respectively. The chemical analysis of dried leaves of Eucalyptus globulus showed high levels of total phenols and total flavonoids contents and exhibited high antioxidant capacity. The results suggest that the leaf aqueous extracts of Eucalyptus globulus had a promising nematicide potential against root lesion nematodes.
Apple rootstock, Pratylenchus vulnus, Eucalyptus globulus, Biological control, Bioactive metabolites
Plant parasitic nematodes are dangerous enemies of several agricultural and horticultural crops. The estimated annual yield loss due to plant parasitic nematodes on major crops of the world is 12.3% . The Pratylenchus species are economically important pests of many crops . The root lesion nematode, Pratylenchus vulnus is recognized as a pathogen of apples and has been shown to be a serious nematode pest causing yield losses in apple orchards in warm mediterranean environments . This species is also pathogenic to almond , peach  and plum .
The plant parasitic nematodes management is commonly based upon chemical treatment. Due to the harmful toxicity in the environment and risks to human health, the use of this type of materials is discussed. Plant derived nematicides could be promoting alternative to pesticides use and fit well to the principles of integrated pest management. Natural products obtained from plants have been used as pest management agents. They served also in some commercial bio pesticides [7,8]. Eucalyptus globulus (Myrtaceae) is an indigenous tree of Australia, widely applied in medicine . The leaves of several species of Eucalyptus have shown biological activities including anti-microbial, fungicidal, insecticidal/insect repellent, herbicidal, acaricidal and nematicidal effects [10,11]. The chemical profile of E. globulus leaves, in particular the essential oil, has widely studied . The leaves of this medicinal tree contain 70% of eucalyptol (1,8-cinéole). A large number of monoterpenoids have been identified, mainly alpha-pinene, s-pinene, δ-limonene, para-cymene, camphene, alphaphellandrene, alphafenchene, γ-terpinene. The aromadendrene and alloaromadendrene are the most found sesquiterpenoids. The monoterpens glycosides (globulisin, cypellocarpin, euglobulin), flavonoid (quercetin, rutin), polyohenols (catechol, cafeic acid, gallol, etc.) are other compounds most found in E. globulus leaves [13-15]. The nematicidal activity of several plants are attributed to these chemical compounds [16-20].
The present study aimed at evaluating the nematicidal effect of dried leaf aqueous extracts of Eucalyptus globules against Pratylenchus vulnus in vitro and in vivo experiments. Additionally, the chemical screening of total phenol and total flavonoïd contents and antioxidant activity of the aqueous extracts were analyzed.
Plant collection and extracts preparation
Eucalyptus leaves were collected from wild trees in the region Oueslatia (center of Tunisia) and dried in the shade at 30 to 40°C for fifteen days. A leaf powder was prepared by crusher the leaves using commercial mortar. To obtain the aqueous extract, 30 g of powdered leaves were placed in glass flasks containing 100 ml of sterilized distilled water. The flasks were placed under an orbital shaker for 48 hours at room temperature (about 25°C). After two days; the water suspension in the flasks was filtered through a what man filter paper N°1 and the filtrate was used directly for in vitro and pot essays. The obtained filtrate was considered as a concentrated solution (100%) and other concentrations (60%, 30%, 15% w/v) were prepared by adding the required amount of sterilized distilled water at the time the bio test and pot experiments were performed.
The preparation of sample and determination of total flavonoids, total phenol, antioxidant activity and HPLC-DAD for phenolic profile were determined as described by Hajji-Hedfi, et al.
In vitro nematicide assay
A suspension of Pratylenchus vulnus specimens provided from roots of MM106 rootstock, after root extraction by double centrifugation method  was used. Fifty individuals of P. vulnus were placed in a plate containing 1 ml of leaf aqueous extract (four concentrations were used; 100%, 60%, 30% and 15%) with ten replicates for each treatment. Plates with distilled water were used as a negative control (0%). The plates were maintained at 25°C in darkness. Then, the effect of the aqueous extracts on P. vulnus viability was checked at 24, 48 and 72 hrs. The response of individuals was observed and the dead nematodes were counted under a stereoscopic microscope. The moved nematodes during 24 hrs were considered alive, whereas those not responding were considered dead. The experiment was repeated twice.
In vivo experiment
A pot experiment was conducted under greenhouse conditions at higher agronomic institute of Chatt-Mariem, Sousse university, Tunisia. Total of 42 trees of the rootstock 'MM106' (one year old each tree) were transplanted each one into 10 kg soil pots filled with a sterilized sandy loam soil (1:1:1v/v). The initial population is determined from all rootstock tree naturally infected with the root lesion nematode (1390 females and 123 males per 1 g of roots), and originated from the region of Oued Melliz of the governorate of Jendouba (north of Tunisia). Four concentrations of leaf aqueous extract of E. globulus were used (100%, 60%, 30% and 15%). Each pot was treated by pipetting 10 ml of each concentration into two holes at 2–4 cm deep around root system. Additional pots were treated with 3 g of Oxamyl G and were used as positive control for comparison. Plants naturally infected and not treated are considered as negative control. All treatments were arranged in a completely randomized design with seven replicates.
After 2, 4 and 6 months of the treatments; a root sampling from each treatment was realized. Taking 10 g of each replication and mixed in order to have a representative sample of each treatment. The P. vulnus root population was determined via root extraction and observation under a stereoscopic microscope. At the end of experiment (after 6 months of the treatments), the growth parameters were measured including: The foliage tree diameter, plant height, trunk diameter, branch number, and number of leaves per branch.
The data were subjected to analysis of variance (ANOVA) using SPSS 20.0 for windows. The bioassays data were analyzed by probit regression to obtain the LD values. Laboratory and pot experiments were repeated twice and the treatment means were compared by the Tukey’s multiple range tests when the F-tests were statistically significant at P<0.05.
Phytochemical analyses of dried leaves composition
The results for flavonoid and total phenolic contents and antioxidant activities measured by DPPH method from aqueous extracts from dried leaves of E. globules showed that the flavonoid and total phenols contents were 14183,28 μg QE/g D.W and 185,45 μg GAE/g D.W, respectively. It was observed that aqueous extracts contained a notable antioxidant activity reached 62,19%.
The overall chemical composition of E. globulus aqueous extract permitted the identification of a number of phenolic acids such as quinic acid and gallic acid, in addition the quercetin, main flavonoids constituent. Nevertheless, other phenolic acids occurred in leaves of E. globulus as Catechin (+), caffeic acid, 4-0-caffeoylquinic 3,4-di-O-caffeoyquinic acidand 1,3-di-O-caffeoyquinic acid but at low rates. The phytochemical analysis of dried leaves showed the high levels of flavonoid and phenol contents and an interesting antioxidant capacity. E. globulus has been reported previously containing biologically active terpenoids, flavonoids, polyphenols and high antioxidant activity [22,23]. Several recent studies have shown that the biocidal activity was attributed to several bioactive molecules revealed the capacity of plant extracts in the prevention and control of plant diseases is due to presence of some natural compounds such sterols, saponins, tannins, flavonoids and alcohols [24-27]. The toxicity of root knot nematodes (M. javanica) treated by F. vulgare and M. spicata were achieved essentially by tanins, saponsides, estragol, phenol . The phenols were reported involved in plant defense against pathogens. The tomato roots immersed in five solutions of phenols showed the decrease of radopholus reniformis infection. The quercetin (flavonoid) inhibited the development of Meloidogyne javanica. The rotenone as flavonoid demonstrated the nematicide activity against Caenorhabditis elegans (Table 1) .
|trans frulic acid||32.50233333|
Table 1. Phytochemical screening of phenolic constituents in dried leaves of E. globulus.
In vitro Effect of E. globulus on root lesion nematode
The efficacy of E. globulus leaf aqueous extracts on P.vulnus mortality, under laboratory conditions, is presented in (Table 2). The results showed that the four concentrations enhanced significantly the P. vulnus mortality as compared with control. After 24 and 48 hours, the undiluted extract (100%) exhibited the highest mortality as compared with the diluted extracts at 60%, 30%and 15%. After 72 hours exposure, both treatment with C1 and C2 exhibited the greatest net mortality (P<0.05). The lethal doses LD50 and LD90 of E. globulus aqueous extracts were 83, 19 μg/ml and 189, 12 μg/ml respectively. The in vitro tests confirmed previous observations on the strong biocidal effect of E. globulus (Figure 1). A crude extract from fruits of E. globulus showed strong antimicrobial activities [30,31]. The results of biocide activity are in line also with the finding of when they reported a high antimicrobial potential for the essential oil of E. globulus when compared with the antibiotics used (ceftriaxone, amykacine and tetracycline). Additionally, a number of investigations have demonstrated acaricide effect against Boophilus microplus  and insecticide activity against several insects genera [33-35].
|Treatment||Mortality% 24 h||Mortality% 48 h||Mortality% 72 h||Mortality% Net|
|Eucalyptus globulus (30 g/100 ml)||62.00 ± 10.32c||85.00 ± 5.27d||96.00 ± 5.16c||95.78 ± 5.46b|
|Eucalyptus globulus (20 g/100 ml)||45.00 ± 17.15b||73.00 ± 6.74c||92.00 ± 6.32c||91.39 ± 7.50b|
|Eucalyptus globulus (10 g/100 ml)||41.00 ± 9.94b||58.00 ± 4.21b||78.00 ± 7.88b||77.17 ± 7.66a|
|Eucalyptus globulus (5 g/100 ml)||43.00 ± 8,2b||59 ±3.16b||78.00 ± 4.21b||77.05 ± 4.05a|
|Control||49.33 ± 0a||72.00 ± 0a||88.67 ± 6.99a||-|
|Values are means ± standard deviation of five replicates. Values in columns followed by the same letter do not differ at P<0.05 according to Tukey's multiple-range test.|
Table 2. Effect in vitro of different concentrations of aqueous extract of Eucalyptus globulus on mortality of P.vulnus.
Effectiveness of aqueous leaves extract of E. globulus against P. vulnus in vivo
After 6 months’ post treatment, the concentration 100% reduced significantly (P<0.05) root lesion nematode development and showed the lowest female and male reduction rate by 84.43% and 91.40% respectively. However, the chemical treatment with Oxamyl G registered important lowest for female and male reduction rates with 98.30% and 100%, respectively. The P. vulnus females were reduced significantly at concentration 100% and concentration 60% but males were only reduced at concentration 100%. In general, the invasion of apple roots by P. vulnus decreased significantly by Oxamyl G followed by concentrations 100%, 60%, 30% and 15% (Table 3).
|Treatments||Foliage tree diameter (cm)||Branch number||Trunk diameter (cm)||Plant height (cm)||Leaves Number/branch|
|Eucalyptus globulus(30 g/100 ml)||10.29a||6.86b||2.71ab||97.14ab||10.43bc|
|Eucalyptus globulus (20 g/100 ml)||9.57a||6.00ab||2.57ab||89.29ab||8.57ab|
|Eucalyptus globulus (10 g/100ml)||8.43a||5.86ab||2.36ab||90.00ab||7.86ab|
|Eucalyptus globulus(5 g/100 ml)||8.21a||5.71ab||2.36ab||90.29ab||7.85ab|
|Oxamyl G (3 g)||17.71b||9.43c||2.93||98.57b||11.71c|
|Values are means ± standard deviation of four replicates. Values in columns followed by the same letter do not differ at P<0.05 according to Tukey's multiple range tests.|
Table 3. Effect in vivo of different concentrations of aqueous leaves extracts of Eucalyptus globulus on apple root stock MM106 growth.
After 2 months, the undiluted aqueous extract, reduced (P<0.05) female and male rate by 56.23% and 60.13%, respectively, compared to others extract concentrations. Oxamyl G enhanced female and male reduction rate respectively by 96.61% and 100%. After 4 months, the concentration 100% showed significantly (P<0.05) female and male reduction rate (74.50% and 82.52% respectively) compared to the chemical treatment female and male reduction rate by 97.51% and 100%, respectively. The potential uses of plant extract to control the root lesion nematode, Pratylenchus spp. have been reported by several researchers . Reported the nematicidal effect of Chenopodium ambrosioides towards Pratylenchus brachyurus. The aqueous extract of Melia azedarah displayed nematicide activity against P. thornei . Furthermore, 75% mortality of P. vulnus juveniles recorded after 96 hours of exposure to 15 μg ml-1 of essential oil from Rosmarinus officinallis .
Oxamyl G enhanced significantly apple growth comparing with control trees and those treated with leaf aqueous extract solutions . The apple trees treated with the highest concentration (undiluted concentration) of leaf aqueous extract showed maximum growth parameters as compared with both concentrations 60% and 30% (P<0.05) (Table 4). Both, chemical treatment and leaf extracts at all concentrations improved significantly the number of leaves per branch compared with control.
|Treatment||Female reduction (%)||Male reduction (%)||Total reduction (%)||Sex ratio|
|Eucalyptus globulus (30 g/100 ml)||84.43 ± 4.45b||91.40 ± 3.76b||84.84 ± 4.24b||1,08b ± 0,06|
|Eucalyptus globulus (20 g/100 ml)||77.97 ± 4.32b||82.84 ± 5.87a||78.30 ± 4.15b||0,97b ± 0,22|
|Eucalyptus globulus (10 g/100 ml)||64.95 ± 9.66a||81.05 ± 5.71a||65.84 ± 9.38a||1,26a ± 0,14|
|Eucalyptus globulus (5 g/100 ml)||63.76 ± 7.66a||78.53 ± 3.10a||64.59 ± 7.26a||1,24a ± 0,12|
|Oxamyl G (3 g)||98.30 ± 0.34c||100 ± 0c||98.40 ± 0.31c||1,01c ± 0,03|
|Values are means ± standard deviation of four replicates. Values in columns followed by the same letter (a, b, c) by colomn do not differ at P <0.05 according to Tukey's multiple range tests.|
Table 4. Effect in vivo of different concentrations of Eucalyptus globules aqueous extract on Pratylenchus vulnus development in apple roots.
Considering the results of total flavonoid and phenol, contents and the antioxidant capacity, the dried leaves of the medicinal plant Eucalyptus globulus contained high levels of phenols and flavonoids and showed a high level of the (DPPH) scavenging. Furthermore, the aqueous extracts of dried leaves showed a significant potential on reducing development of the root lesion nematode Pratylenchus vulnus in vitro and in vivo. These findings provide scientific evidence to support the value of medicinal plants for pest management and indicate that E. globulus could be a promising source for potential biological control agent. Further work should be realized on the identification; isolation of the spcific compounds from the leaf extracts which are responsible for the higher nematicidal action.
Thank you to all people helps me to achieve this manuscript (specially the technical team of the laboratory of nematology in the higher agronomic Institute of Chott Mariem (ISA CM).
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