Abstract: Background and Objective: Pesticidal plants have become increasingly popular among small scale farmers in Kenya. Little is known about the efficacy of the non-volatile bioactive compounds of this species. There exist gaps such as the chemical compounds responsible for pest repellency and the right concentration of the botanical extracts. This study, therefore, determined the non-volatile bioactive compounds and evaluated the efficacy of three concentrations of L. javanica complex in control of aphids of Vigna unguiculata. Materials and Methods: Crushed powder of L. javanica leaves which were collected from different localities were used in the preparation of crude extracts, which were then screened for the presence of the bioactive compounds. Three prepared concentrations of 10, 5 and 1% were sprayed on cowpea plants planted on four blocks which had been replicated four times. Cowpea aphids were enumerated according to the categorical indices prior to and after spraying. Synthetic pesticide atom (Osho) and water plus soap were used as positive and negative controls, respectively. Results: Lippia javanica was found to be rich in a variety of non-volatile compounds namely: phenols, flavonoids, tannins, alkaloids, cardiac glycosides and terpenoids that perhaps exhibit pesticidal effects on cowpea aphids. However, phenolic glycosides, resins and polyuronides were absent. Application of the extracts irrespective of concentrations significantly suppressed aphid population at p<0.05. It was notable that aphid suppression increased with increasing concentration. Consequently, the mixture of L. javanica extracts at 10% outperformed 1 and 5% extract concentration. In addition, the 10% extract concentration favorably competed with the synthetic insecticide (Atom, Osho Syngenta). Conclusion: The findings from this study showed that Lippia javanica possessed a wide range of phytochemical compounds; also, the 10% botanical extract was found to be comparable to the synthetic pesticide already in use for control of cowpea aphids.
INTRODUCTION
Lippia javanica is a multi-branched woody shrub of the tribe verbenae of the Verbenaceae family. It is a strongly fragrant medicinal indigenous plant that naturally occurs in eastern and southern tropical Africa1. Its essential oil composition have been investigated widely. Viljoen et al.2 reported that myrcenone, myrcene and (E)-and (Z)-tagetenone as among the major constituents of the essential oils of L. javanica. Other components were found to be caryophyllene, linalool and p-cymene2. The essential compounds were found to vary sharply even with the samples taken from the same location. Geranial and neral chemotypes were found to occur in Tanzania2. However, most research on the chemical composition of L. javanica focuses on volatile essential oils. The non-volatile phytochemical compounds have been partially evaluated. Even though Maroyi3 determined non-volatile secondary metabolites such as alkaloids, amino acids, flavonoids, iridoids and triterpenes, it is worth to note that a wide range of other non-volatile compounds were neglected. Previous studies on L. javanica showed it to be acaricidal used by smallholder farmers against cattle ticks4. Accordingly, it has been used as a cheap supplement in controlling cattle ticks in southern Africa. Other applications of the extracts include management of microbial infections like coughs or colds in humans. It also exhibited wound healing properties thus used to treat skin infections. According to Maroyi3 the previously mentioned non-volatile compounds which were isolated from L. javanica and volatile oils are responsible for antimicrobial properties. Bio-active compounds are believed to act with synergic effect to deter microbes and pests. Pests, therefore, find it difficult to develop resistance to them, thus they should be further evaluated. For instance, the right concentration for the botanical extracts to be used as natural pesticides is unknown. Plants with pesticidal properties have been investigated for centuries as alternatives to synthetics. However, little progress has been made to develop new effective products5. Although research on pesticidal plants is increasing, it is failing to address gaps in our knowledge that constrain their adoption6. Studies concerning pesticidal efficacy on L. javanica have been partially addressed while the findings are unpublished. In the southern Africa region, L. javanica is known to demonstrate variation in efficacy. There was a need to investigate if any variation in terms of chemical compounds and efficacy of the different concentrations on aphids existed among L. javanica complex species. Extracts from this aforementioned species complex perhaps may play a significant role as pest repellants.
MATERIALS AND METHODS
Collection and processing of the L. javanica plant materials: Fresh aerial parts of L. javanica plant were collected in the floral areas of Kenya where it is known to occur in June/July 2016 growing season. The plant materials were stored in dark bags and transported to Maseno University botany laboratory. Immediately, the L. javanica materials were cleaned and air-dried under the shade and then crushed into a fine powder using Kika Werke M20 grinder.
Qualitative test for the phytochemical compounds: The extracts were screened for presence of non-volatile phytochemical constituents using standard methods as reported by Nalubenga et al.7. To test for the presence of alkaloids, Mayer’s reagent was used while cardiac glycosides were detected by Keller-Killiani test. Whereas the foam test detected presence of saponins. Shibata’s reaction was used to confirm presence of flavonoids. Tannins were detected using Braemer’s test. Coumarins were tested by evaporating ether extract to dryness and dissolving the residue by heating in 2 mL of water and they were confirmed if 0.5 mL of 10% ammonia solution was added and a blue or green fluorescence under ultraviolet light developed. To detect for the presence of polyuronides, about 2 mL of aqueous extract were added dropwise to 10 mL of ethanol. Formation of a thick precipitate indicated presence of polyuronides. The formed precipitate was separated off and washed away with ethanol and stained with methylene blue and occurrence of violet color further denoted presence of polyuronides.
Study site for the field experiment: Pesticidal efficacy study was undertaken in Makueni County (Nzouni, village). The study was conducted from November 2015 to February, 2016 growing season on Mrs. Patricia Mukai’s farm (S 02 00.877 E 37.862569, 920 m above sea level). The soil type was a mixture of red sandy and clay black cotton soils8.
Plant materials collection and processing: Fresh leaves of L. javanica were collected from different localities around Kenya where the species occurs naturally. Voucher specimens and Geographical Positioning Systems (GPS) coordinates were lodged at the East Africa Herbarium Nairobi National Museum. Leaves were dried under shade for a week and then ground into fine powder using a grinder.
Field preparation and cowpea planting: The field was disc harrowed and ridged prior to planting. The common cowpea (Vigna unguiculata) seeds used for planting were of the variety Katumani KVU 27-1. They were obtained directly from the breeder at Kenya Agricultural and Livestock Research Organization (KALRO). The seeds were planted at a spacing of 50 cm between rows and 20 cm within rows in 3×4 m plots which were 1 m apart. Three seeds were planted per hole and then thinned to two plants one week after germination. The experimental layout was a randomized complete block design and the treatments were replicated on 4 blocks, all within the same field location.
Preparation of the three concentrations of the botanical extracts and field treatments: To determine effective pesticidal concentration three different botanical extracts 10, 5 and 1% w/v were prepared. As extraction was carried out in water, a second variable of 0.1% soap was added. The extracts were kept in sealed plastic buckets to extract at ambient temperature (20±5°C) overnight. Thus, there were 5 treatments 1, 5 and 10% plus positive and negative controls. Each was replicated four times, thus giving 20 blocks. Extracts were kept in 10 L buckets with tight lids in the shade and filtered through a fine cloth to remove all plant materials that may inadvertently clog the sprayer. The positive control in the trial was a synthetic pesticide Atom (Osho, Syngenta). The number of aphids before the treatments were counted and found to have an average of eight. All treatments were sprayed throughout the growing season at an interval of 7 days starting one week after cowpea plant emergence.
Sampling of aphid infestation: All assessments were carried out the day before treatments were to be sprayed following the modified method6. The target insect pests to be evaluated were cowpea aphids. Three inner rows from each plot were selected for sampling. Due to often very high numbers, a categorical index was used to assess aphid abundance according to the modified method6:
| • | 0 = None |
| • | 1 = A few scattered individuals |
| • | 2 = A few isolated colonies |
| • | 3 = Several isolated colonies |
| • | 4 = Large isolated colonies |
| • | 5 = Large continuous colonies |
The severity or degree of infestation in each infested plant was assessed by scoring the extent of damage using scoring grades6. After spraying with all the extracts, a similar procedure of sampling was conducted again the following day in the morning. The difference was taken to be the number of aphids suppressed where it was recorded:
| • | 0 = No damage |
| • | 1 = Damage up to 25% |
| • | 2 = Damage from 26-50% |
| • | 3 = Damage from 51-75% |
| • | 4 = Damage more than 75% |
Data analysis: Differences among treatments in the number of aphids and severity of damage were assessed by one-way analysis of variance (ANOVA). Means were separated by Least Significant Difference (LSD) test at 5% probability level. Analyses were performed in STATA data analytical package.
RESULTS
Phytochemical composition of L. javanica: The study revealed that the extracts from the leaves of L. javanica had a variety of phytochemical groups (Table 1). Out of the 13 non-volatile phytochemical compounds screened, eleven were present.
| Table 1: | Results for phytochemical screening of bioactive compounds in the L. javanica for 10 accessions of L. javanica |
| ++: Highly present, +: Fairly present and -: Absence of phytochemical. Sondu (0019; 0020), Kedong (0025; 0029), Nyahururu (0035; 0036), Maralal (0037A; 0037B), Narok (0038; 0039) | |
| Fig. 1: | Aphid abundance (number of aphids repelled) of cowpea aphids repelled after being sprayed with extracts of three conc. 1, 5 and 10% of L. javanica plant and positive/negative controls treatments after 10 weeks of treatment |
| Different alphabet letters (a, b,c and d) indicate significant differences | |
| Fig. 2: | Effect of the three conc. 1, 5 and 10% of L. javanica plant extracts and positive/negative controls treatments on severity or degree of aphid infestation (damage) after 10 weeks of treatment |
| Different alphabet letters (a, b,c and d) indicate significant differences | |
The three compound groups namely resins, phenolic glycosides and polyuronides were confirmed to be absent. Flavonoids, terpenoids, phenols, cardiac glycosides, saponins and alkaloids were present in high amounts (++). Flavones, anthraquinones, coumarins, anthocyanins and tannins occurred in fairly present amounts (+) irrespective of the locality.
Efficacy of the extracts of L. javanica in control of aphids of V. unguiculata: There were significant differences in efficacy of the extracts of L. javanica among the treatments where, the number of pests suppressed increased with increasing concentration of the treatments (Fig. 1 and 2) (F = 184.543 and p<0.001). However, there were no significant differences between the 10% extract level and the synthetic pesticide (positive control) (F = 184.543 and p<0.001) implying that the 10% concentration competed favorably with the synthetic pesticide (Atom, Osho from Syngenta). Also, it was notable that aphid numbers increased in the plots sprayed with water and soap solution (negative control).
Similarly, the severity or aphid infestation was highest where water and soap solution was applied. Aphid infestation decreased with increasing concentration of the extract. The results for analysis of variance (ANOVA) in the comparison of aphid infestation mean across the five treatment showed significant differences among the treatments (F = 194.143 and p<0.001). Aphid infestation was, however, least in the 10% and the synthetic pesticide (positive control), where there was no significant difference between the two treatments (F = 194.143 and p<0.001.
DISCUSSION
Lippia javanica was found to possess flavonoids, flavones, terpenoids, phenols, cardiac glycosides, saponins, alkaloids, anthraquinones, coumarins, anthocyanins and tannins. However, polyuronides, resins and phenolic glycosides were absent implying that the aforementioned species did not demonstrate dramatic variation in phytochemical compounds. Collection of the L. javanica materials in the same season and time was attributed to be the cause of lack of the variation. This highly aromatic plant has been reported to display great variations in phytochemical compounds9. Furthermore, Viljoen9 noted that the dramatic variations in the essential and non-essential compounds was due to regional and different harvesting time. Nevertheless, Kamanula et al.10 found that the qualitative phytochemical compounds did not vary among the different accessions of L. javanica which agrees with the results of this study. Surprisingly, phenolic glycosides were absent in this present study contrary to Endris et al.11 findings. Also, essential oils were reported to vary with mycene, mycenone, linalool, caryophyllene and p-cymene among the chemotypes isolated from L. javanica10,12. A study by Sahreen et al.13 found out that the non-essential greatly varied in Rumex hastatus L. a situation that was attributed to different harvesting time. It is worth noting that Endris et al.11 also had similar findings in the L. javanica from Ethiopia. According to Nalubenga et al.7, a number of plants contained chemical components that have been reported to be biologically active. These plants, therefore, have various parts such as; leaves, roots, rhizomes, stems, barks, flowers, fruits, grains or seeds, employed in the control or treatment of various disease conditions14. Leaves of L. javanica have a wide variety of the so-called classic nutrients, such as minerals, carbohydrates, proteins, fats and vitamins11.
According to Maroyi3, total phenolic compounds, tannins and minerals were isolated from L. javanica. Another study by Madzimure et al.15 reported that alkaloids, flavonoids, iridoids, triterpenes and amino acids were confirmed to occur in L. javanica. As a matter of fact, alanine, asparagine and arginine were the amino acids detected16. Basic alkaloids, flavonoids, flavones were present in L. javanica3. Nevertheless, these compounds were found to vary in quantity. Lippia javanica leaf extracts chemical composition surprisingly varies within and between populations Madzimure et al.15 owing to edaphic and climatic factors17. While phenolic glycosides were reported in high amounts in L. javanica, Dlamini16 found out that they were completely absent. The fluctuation in some of the compounds was attributed to different times in harvesting, the maturity stage and season.
This study demonstrated that L. javanica possessed pesticidal effects on aphids that affect cowpea plants (Fig. 1). This scenario meant that the L. javanica was rich in compounds, which include alkaloids, flavonoids, flavones and terpenoids which were reported to exhibit anti-feedant and repellency and toxicity on insects. According to Stevenson et al.5, the botanical pesticides of L. javanica acted in a synergistic effect to deter small bodied pests including rape spider mites and aphids. Several constituents in the volatile component have been identified in L. javanica. The major component camphor which has insecticidal properties6. Camphor occurred with other minor components including camphene, α-pinene, eucalyptol, Z and E α-terpineol, linalool, cymene, thymol, 2-carene, caryophyllene and α-cubebene and may account for the biological activity of plant species in this study18. Other potential biologically active components present in L. javanica were mono and sesquiterpenes i.e., perialdhyde in the essential oils, this compound is highly toxic through contact with insects. The efficacy of botanical insecticides on aphid pests was not fully effective. Some aphid pests had chances of survival perhaps through re-infection and behavioural mechanisms. Similar trends of the sought have been observed, like a study by Stevenson et al.5 found out that rape aphid pests affecting tomato developed resistance against the botanical extract of L. javanica and Vernonia amygdalina. It is also possible that the active ingredients of pesticidal plants were quickly photodegraded upon their application and, therefore, reducing efficacy since they were sensitive to light19. Dissolving of the plant powder in the water overnight may have not achieved total extraction of all the pesticidal compounds. Especially non-polar compounds perhaps were not fully dissolved even though soap was used in the process. According to Mkenda et al.6, the method of extracting the active ingredients also caused variations in concentration of the potent substances hence affecting efficacy. Chances may have been that the survival of the aphid population was caused by new infestation from neighboring cowpea crop areas20. The extracts of L. javanica were able to suppress the aphids’ abundance and minimized cowpea damage below their economical threshold. The reduced number of aphids and mites was due to extracts’ repellency, toxicity and anti-feedant effects since they contained alkaloids and other constituents with pesticidal properties19. These findings were in agreement with the findings of Mkenda et al.6, who found out higher concentration of L. javanica botanical extract (20%) was comparable to the synthetic pesticides in reducing damage caused by foliage beetle. Vernonia amygdalina and L. javanica were the most effective plant species treatments to reduce damage caused by aphids6.
CONCLUSION
Lippia javanica was found to be rich in phytochemical compounds such as alkaloids, tannins, flavonoids, flavones, cardiac glycosides, phenols, proteins, saponins and terpenoids, which may play a vital role in repelling aphids. These compounds make the species a potential pesticidal plant. Notably, phenolic glycosides were absent contrary to the findings of other researchers. There was no significant difference between the 10% extract concentration of L. javanica and synthetic pesticides treatments. Therefore, the 10% extract of the pesticidal L. javanica plant can be utilized as an alternative option for controlling field cowpea aphids instead of 5% which is relatively weak an 20% requires more powder and tiresome to prepare.
SIGNIFICANT STATEMENT
This study discovered that the 10% L. javanica botanical extract is the most suitable in controlling aphids. This study will help the researcher to uncover the critical areas of pesticidal plants use that many researchers were not able to explore. Thus a new theory on the right concentration of botanical extracts in controlling soft-bodied field pests on crops may be arrived at.
ACKNOWLEDGMENT
We thank the technical field assistance provided by Mrs. Mukai. We also acknowledge the facilitation by Optimization of Pesticidal Plants Outreach and Innovation programme.