Bio-Nematicidal Effect of Moringa oleifera on Citrus Nematode Tylenchulus semipenetrans

Citrus is the leading fruit crop of Pakistan. It is highly nutritive, and rich in Vitamin A and C, organic acids, sugars, amino acids, and carotenoids (Cohn and Duncan, 1990). Several biotic and abiotic factors have contributed to the low yield and poor quality of citrus in Pakistan. Tylenchulus semipenetrans is one of the most prevalent pests of citrus which is associated with the citrus slow decline (CSD) and severely limits the yield (Pervez et al., 2003). CSD is characterized by poor root development, and abnormal or sick appearance of plants with significantly low yield (Iqbal et al., 2006; Khanzada et al., 2008). It mainly invades though young feeder roots and the infection is aggravated by limited water availability and soil stress (Duncan and Noling, 1987).

Several management strategies have been used to control this nematode (Nasir et al., 2021). Among them the chemical pesticides are most widely used. However, nematicides are expensive and toxic to the environment (Wang et al., 2002). Moreover, the pathogens may develop resistance to the chemicals (Okigbo and Nmeka, 2004; Carvalho, 2004). The use of bio-pesticides of botanical origin is an eco-friendly way to manage CSD (Siddiqui et al., 2005). Botanical pesticides are biodegradable and least toxic to human health and the environment. Several antifungal plant extracts have been tested for their nematicidal effect against nematodes (Khan et al., 2019; Aminisarteshnizi, 2021).

Moringa oleifera Lam (Moringaceae) is native to the western and sub-Himalayan region, India, Pakistan, Asia, Africa, and Arabia, cultivated in the Philippines, Cambodia, Central, North and South America, and Caribbean Islands (Morton, 1991). It is a fast-growing and drought-resistant deciduous tree. M. oleifera leaves and seeds possess antimicrobial properties against several pests of fruit crops (Fahey et al., 2005). M. oleifera contains antimicrobial compounds such as saponin, tannins, alkaloids, phenols, steroids, and reducing sugars. These phytochemicals have bio-nematicidal potential (Izuogu and Oyedunmade, 2009). M. oleifera extract reduced nematode population in mung bean, cowpea, and urdbean and also enhanced plant yield (Devi, 2002). Similarly, Murslain et al. (2014) recorded significant mortality of M. javanica on eggplant by the application of M. oleifera in combination with Trichoderma harzianum. Claudius-Cole et al. (2010) and Izuogu et al. (2013) also recorded the mortality of M. incognita in cowpea and maize, respectively by M. oleifera extracts. M. oleifera boosts crop yield by decreasing the nematode population.

The project was undertaken to evaluate the nematicidal efficacy of M. oleifera extracts to cause mortality and reducing the invasion and development of T. semipenetrans in citrus.

T. semipenetrans was isolated from root and soil samples collected from infected citrus trees following Whitehead and Hemming tray method (Whitehead and Hemming, 1965) and Baermann funnel method (McKenry and Roberts, 1985). T. semipenetrans was mass cultured on Citrus jambhiri (Rough Lemon). Six-week-old citrus seedlings were transferred to earthen pots containing sterilized sandy loam soil. The plants were inoculated with 1000 J2/pot of T. semipenetrans and kept in the greenhouse. The plants were watered once a week.

M. oleifera sample was taken from the botanical garden University of Agriculture, Faisalabad, Pakistan. Leaves were washed, surface sterilized with sodium hypochlorite and 95% absolute ethanol. The leaves were oven-dried at 70 ºC for 2 days. To prepare fresh and dry leaf aqueous extracts 10g of fresh and dry leaves were ground separately in 100mL distilled water, while, methanolic extracts and ethanolic extracts were prepared by grinding 10g of fresh and dry leaves in methanol and ethanol (Adegbite and Adesiyan, 2005). The extracts were filtered though Whatman No. 1 filter paper. The initial concentration was 10% and it was maintained as standard concentration “S”. The S concentration was diluted up to 25% and 50% and maintained as S/2 and S/4 concentrations, respectively. The mortality of T. semipenetrans was assessed by pouring 5mL botanical extract from each concentration, S, S/2, and S/4 in sterilized Petri dishes 5cm in diameter. T. semipenetrans inoculum 1mL containing 25 freshly hatched J2’s was pipetted out in the Petri dishes. Control treatment contained only 1mL nematode inoculum suspension. The Petri dishes were incubated at 25-28 oC. Each treatment consisted of thee replicates. The juvenile mortality was assessed at 24, 48, and 72 h. Juvenile mortality was calculated by the formula given:

Effect of M. oleifera extracts on invasion and development of T. semipenetrans

Air-dried sieved soil was treated with “S” concentration of M. oleifera extract and distributed into 250 mL volume sterilized plastic pots. The soil was distributed in the pots and after 2 days 6-weeks old citrus plants were transplanted into these pots and kept in a growth chamber at 25 ºC for 12 weeks. In the control treatment, the soil was kept untreated. After 1 week of transplanting the plants were inoculated with 1000 freshly hatched J2’s of T. semipenetrans. The experiment consisted of a set of 4 treatments and each treatment was replicated five times. Data on the development stages of T. semipenetrans was recorded at 1, 6, and 12-week. The total number of J2, J3, J4, immature females and mature females of T. semipenetrans were recorded.

To study the in vivo protective and curative effect of M. oleifera extracts on the invasion and development of T. semipenetrants six-months old citrus plants were transferred to earthen pots containing sterilized soil. The most effective concentration of aqueous, methanolic, and ethanolic M. oleifera extract from in vitro study was selected for this study. In the case of protective treatment 50mL, M. oleifera extract was sprayed on each plant though a calibrated sprayer. Plants were inoculated with freshly hatched 1000 J2’s of T. semipenetrans. The inoculum was directly applied to the soil around the root zone of the plants. In the case of curative treatment 50mL M. oleifera extract was sprayed one day after inoculation with 1000 freshly hatched J2s of T. semipenetrans. The plants were kept in the greenhouse at 25 ºC for 8 weeks. Reproduction parameters of T. semipenetrans number of juveniles/g root system, number of females/g root system, and number of juveniles/100 mL soil were recorded by following the formula given below:

Plant growth parameters plant height (cm), number of leaves, and root length (cm) were also recorded after 8 weeks.

The experimental design was completely randomized. The data were subjected to Tukey’s highly significant difference test (HSD) after ANOVA analysis. Data were analyzed using the statistical software Statistix (Ver, 8.1). Significant interactions were used to interpret the results.

In vitro, the effect of aqueous, methanolic, and ethanolic fresh and dry leaf extract of M. juvenile on juvenile mortality of T. semipenetrans indicated that plant extract treatment, concentration, time and their interaction had a highly significant effect on juvenile mortality (Table I). Both fresh and dry leaf extracts of M. oleifera showed highly significant juvenile mortality of T. semipenetrans. Extracts from all types of media ethanol, methanol, or water showed complete juvenile mortality of T. semipenetrans after 72-h. The lowest juvenile mortality was observed at 24-h which increased significantly after 72-h. The highest mortality was produced at “S” concentration, while, mortality was least by diluting the “S” concentration up to 50% “S/4” (Table II).

Table I. Interaction effect of M. oleifera extracts, their concentration and time of exposure on in vitro juvenile mortality (%) of T. semipenetrans.

*, significant, **, ***, highly significant, ns=non-significant at P=0.05, ANOVA.

Values are mean of 5 replicates. Values in the columns with same letters are not significantly different from each other, analyzed by Tukey’s HSD test at P=0.05.

The interaction of plant extracts and weeks on invasion and development had a highly significant effect on all developmental stages of T. semipenetrans J2, J3, J4, immature females, and mature females. Plant extracts, weeks, and their interaction affected the invasion and development of T. semipenetrans (Table III). Ethanolic M. oleifera extract showed the lowest invasion of all developmental stages of T. semipenetrans J2, J3, J4, mature females, and immature females. At week 1, only the J2 stage was observed. At week 6, J3 and J4 stages were observed, while, at week 12 J3, immature females and mature females were observed (Table IV).

For statistical details, see Table I.

For statistical details, see Table II.

Growth parameters of plant height, root length, and the number of leaves were also taken after checking the effect of M. oleifera extracts on the invasion and development of T. semipenetrans. Methanolic M. oleifera extract showed maximum plant height, root length, and the number of leaves after control, while, infected control showed the lowest growth of C. jambhiri (Table V).

For statistical details, see Table II.

Protective vs curative effect on growth parameters of rough lemon C. jambhiri and reproductive parameters of T. semipenetrans

In vivo the interaction effect of curative and protective application of M. oleifera extracts on growth and reproductive parameters was assessed. Plant extracts and type of application affected plant height, root length, number of leaves, juveniles/100mL of soil, juveniles/g root, and females/g root, while, their interaction affected the number of leaves, juveniles/100mL soil, and females/g root. The interaction of treatment and time did not affect plant height, root length, and juveniles/g root (Table VI).

Table VI. Interaction effect of protective vs curative effect of M. oleifera extracts on growth parameters of rough lemon C. jambhiri and reproductive parameters of T. semipenetrans.

For statistical details, see Table I.

The effect of the protective and curative application of M. oleifera extracts on the reproduction and growth of C. jambhiri was compared. Protective treatment by methanolic M. oleifera extracts was most effective to decrease the reproduction rate of T. semipenetrans i.e., juveniles/100mL soil, females/g root, and juveniles/g root followed by ethanolic and aqueous extracts (Table VII). Protective application of methanolic extract also showed maximum plant growth. Root length, plant height, and the number of leaves were maximum in plants subjected to the protective treatment of methanolic extract followed by ethanolic and aqueous extracts (Table VIII). Protective application of all plant extracts was more effective to decrease the reproduction of T. semipenetrans and increasing the growth of plants. An inverse relation between the reproduction of nematodes and plant growth was observed. It can be concluded that M. oleifera extracts had the potential to decrease the development and reproduction of T. semipenetrans, thereby boosting plant growth.

Table VII. Effect of protective vs curative application of M. oleifera extracts on reproductive parameters of T. semipenetrans.

For statistical details, see Table II.

Table VIII. Effect of protective vs curative application of M. oleifera extracts on growth parameters of rough lemon C. jambhiri.

For statistical details, see Table II.

Plant-parasitic nematodes are an economically important group of soil-borne pathogens that are attempted to control though resistant germplasm, cultural practices, nematicides, and botanical extracts. Nematodes are a vast group of multicellular animals on earth representing 2/3rd of the total fauna (Khan, 1993). Hence, the magnitude of damage they cause to crops is immense. T. semipenetrans is a theatening pest of citrus around the globe. The root injuries inflicted by citrus nematodes can increase the risk of damage by other pathogens. Although, nematicides are effective to control nematodes, however, most pesticides have been banned due to the high risk to human health and the environment (Veremis and Roberts, 1996). The biological control of nematodes using antagonistic microbes and botanical extracts has become one of the most reliable alternatives to nematicides (Stirling, 1991).

The in vitro and in vivo application of M. oleifera extracts caused significant larval mortality of T. semipenetrans. The results of the present investigation showed that methanolic extract of M. oleifera inhibited the development of nematodes under field conditions, which is in line with the previous findings (Hafeez et al., 2000; Sharon et al., 2001; Claudius-Cole et al., 2010). M. oleifera contains toxic compounds such as saponin, tannins, alkaloids, steroids, and reducing sugars. The mortality of nematodes is due to the antimicrobial effect of these compounds. These basic phytochemicals confer a bio-nematicidal effect (Fatoki and Fawole, 2000; Izuogu and Oyedunmade, 2009). Some of these phytochemicals such as tannins and saponins are reported to possess a nematicidal effect disrupting biological membranes, thereby, facilitating the penetration of toxic compounds (Agrios, 2005). M. oleifera extract has been used against several plant-parasitic nematodes and they didn’t show any phytotoxic effect which indicates that they are safe for use (Claudius-Cole et al., 2010; Izuogo et al., 2013; Sowley et al., 2014).

M. oleifera extracts reduced the penetration and reproduction of nematodes. Therefore, it has been concluded that methanolic extract of M. oleifera proved to be very effective in suppressing the development and reproduction of T. semipenetrans under field conditions. In the present investigation not only, the reproduction of T. semipenetrans was reduced but plant growth was also enhanced. Ahmad et al. (2004) reported that aqueous extracts of Azadirachta indica, Datura alba, and Calotropis procera caused larval mortality and reduced the multiplication and reproduction of T. semipenetrans on C. jambhiri. The protective treatment of M. oleifera extracts was more effective than curative. The protective effect may be due to the presence of inhibitory chemical compounds in M. oleifera extracts. Shawky and Al-Ghonaimy (2015) support the present investigation, where, they stated the effect of 3 plant extracts Origanum majorana, Tagetes erecta, and Eucalyptus globules on T. semipenetrans and reported a reduced population of nematode and enhanced growth of the plants. The lowest number of females and J2 was observed in the protective application of M. oleifera extract. The growth parameters were also enhanced by the application of M. oleifera. Devi (2002) evaluated the effect of Ocimum sanctum, Azadirachta indica, Myristica malabarica, M. oleifera, Mangifera indica, and Acacia Arabica on mung bean, cowpea, and urdbean against nematode infection and observed that they not only reduced nematode population but also enhanced plant yield. The growth-promoting effect of plant extracts has been also reported previously by Awan et al. (1992) and Murslain et al. (2014), who, observed decreased population of nematodes and better growth of the plant. The application of M. oleifera extracts decreased the reproduction rate of nematodes and promoted the growth of plants. T. semipenetrans affects the root system directly, thereby, hindering the uptake of minerals and water that ultimately results in weaker plants. The decline in nematode reproduction decreased the disease attack that aided in promoting plant growth.

It can be concluded that the use of M. oleifera extract can be helpful to control T. semipenetrans. The use of botanical extracts will probably be an economical and effective approach. The active components of M. oleifera extract if isolated in pure form and the bulk amount will form a base for the development of bionematicides. Hence, the future direction of the study will be to test the efficacy of M. oleifera extracts on a large scale and study the active ingredients involved in the inhibitory effect.

The authors would like to acknowledge the Deanship of Graduate Studies and Scientific Research of Taif University, Saudi Arabia, for funding this work.

This research was funded by the Deanship of Graduate Studies and Scientific Research, Taif University, Saudi Arabia.

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

The authors have declared no conflict of interest.