Summary
The legume cavalcade, Centrosema pascuorum, is used extensively as a cover crop and as a component of conservation agriculture systems. It is also an attractive rotation or cover crop for the management of root-knot nematodes (RKN; Meloidogyne spp.) as it is a non-host. RKN are persistent pests that are well known to be difficult to control. However, the mechanisms governing the non-host status of cavalcade is unknown. The current study established that cavalcade leaves are toxic to RKN as either aqueous extracts or soil amendments. Bioassays conducted using Meloidogyne javanica showed that a 90% concentration of aqueous extract derived from 1-month-old cavalcade leaves (89 mg crude extract ml−1) suppressed nematode hatch (82.9%) and killed infective second-stage juveniles of M. javanica (85.3%). Soil amendments with 1% (w/w) of 1-month-old cavalcade leaves (0.99 mg crude extract g−1 soil) also provided effective control of M. javanica in the glasshouse on okra. One-month-old leaves appeared more effective than 2- or 3-month-old leaves. The soil amendments had no adverse phytotoxic effect on okra seed germination. Our study demonstrates the potential for using cavalcade leaves or extracts to manage RKN. This may be due to the nematicidal activity of the various compounds in the leaves, such as flavonoids, phenols and terpenoids, which should be further assessed.
Root knot nematodes (RKN), Meloidogyne spp., are intractable, soil-borne pests with few management options available to farmers. Among nematode pests, RKN are listed as the most important, while they are also viewed as amongst the most important biotic threats to crop production in the tropics (Jones et al., 2013; Coyne et al., 2018). So far, over 100 known Meloidogyne species are associated with damage to horticultural and ornamental crops (Brito et al., 2010; Lopes-Caitar et al., 2019; Rusinque et al., 2022). The most damaging species are M. arenaria, M. incognita, M. javanica and M. hapla (Moens et al., 2009) but the list has recently included M. enterolobii (Philbrick et al., 2020). A key feature of these species is their polyphagous nature and extensive host ranges. Meloidogyne javanica, for example, has a worldwide distribution, with over 770 plant species from a wide range of crop types listed as good hosts (Goodey et al., 1965; CABI, 2022). In vegetable production, RKN cause 15-85% yield losses, depending on nematode densities and crop (Tuncsoy, 2021). The damage threshold of M. javanica on bell pepper is 1.8 eggs and juveniles (g soil)−1 at planting (Moosavi et al., 2015). In Kenya, M. javanica has been detected in several crops, such as bean (Ngundo & Taylor, 1974), cassava (Coyne et al., 2006), chickpea (Maina, 2016), famine weed (Chitambo et al., 2018), passion fruit (Kanyagia, 1993), pigeon pea (Sharma et al., 1993), pineapple (Kiriga et al., 2021), potato (Gichure & Ondieki, 1984), pyrethrum (Parlevliet, 1971), sesban (Desaeger & Rao, 2000), sweet potato (Njuguna & Bridge, 1998) and tomato (Birithia et al., 2012).
The sustainable management of RKN is difficult to achieve, partly due to their highly polyphagous nature, and partly due to the unspecific above-ground symptoms they are often overlooked or misdiagnosed. Although various management options are available, a combination of methods is undoubtedly necessary for their management, and use of the methods is also dependent on local conditions and circumstances (Viaene et al., 2006; Coyne et al., 2009; Wendimu, 2021). Whilst there are various management methods available, there is a need to update, adapt and add constantly to the armoury against RKN.
Numerous studies have demonstrated the successful use of plant extracts to control RKN. For instance, aqueous extracts of Vetiver zizanioides rendered 70% of M. incognita second-stage juveniles (J2) dead (Jindapunnapat et al., 2018); aqueous extracts of Mucuna pruriens, Crotalaria spectabilis and Dolichos lablab killed 44, 33 and 41% of J2 of Meloidogyne spp., respectively (Osei et al., 2010), and aqueous extracts of Crotalaria spp. resulted in 41-100% paralysis of M. incognita, M. javanica and M. enterolobii J2 after 48 h exposure (Jourand et al., 2004). Besides the use of extracts, leguminous soil amendments or planting as cover crops is being increasingly adopted to suppress RKN. Kankam et al. (2015) and Patel & Dhillon (2017) demonstrated that the application of sunn hemp (C. juncea) as a soil amendment decreased M. incognita densities by up to 94%. Jindapunnapat et al. (2019) found that incorporating 10% fresh V. zizanioides into the soil reduced M. incognita infection of cucumber by 46-67%. In Cambodia, C. pascuorum and Stylosanthes guianensis ‘Nina’ have been used as cover crops under conservation agriculture in rice fields, resulting in a decrease of Hirschmanneilla spp. and M. graminicola root densities (Beesa et al., 2021; Masson et al., 2022).
The nematicidal compounds from numerous plant species have received extensive attention to date (e.g., Chitwood, 1992; Chen & Song, 2021) with numerous identified, viz. polythienyls, alkaloids, acetylenes, quassinoids, fatty acids and derivatives, miscellaneous phenolics, flavonoids, terpenoids, post-infectional compounds, and miscellaneous ketones and aldehydes. Wang et al. (2002) reported that C. juncea generated allelochemicals such as pyrrolizidine, alkaloids and monocrotaline that are toxic to Meloidogyne spp. Yang et al. (2015) also analysed compounds from Camellia spp. seed cake extracts by GC-MS and found that 4-methylphenol exhibited high nematicidal activity. In addition, Rocha et al. (2017) determined the nematicidal compounds from aqueous crude extracts of Canavalia ensiformis as d-glucose, l-canavanine, xanthotoxin, cis-aconitic acid, trans-aconitic acid, malic acid, citric acid, palmitic acid and S-carboxymethylcysteine.
Cavalcade (Centrosema pascuorum) is a leguminous crop belonging to the Family Fabaceae (Clements, 1984) originating from tropical South and Central America (Clements, 1984). Cavalcades are mainly used as a hay crop to feed cattle (Cameron, 2005). Recently, Suong et al. (2019) and Beesa et al. (2021) demonstrated the yield benefits to rice when simultaneously planted with cavalcade and S. guianensis in Cambodia. The improved rice yields likely resulted from the higher nutrient and organic carbon availability, but also possibly from the nematode management, as cavalcade has been shown to act as a non-host to M. graminicola (Suong et al., 2019). However, no further information appears to exist on the nematode management potential of cavalcade. Therefore, the current study was conducted to evaluate using cavalcade leaves and aqueous extracts as soil amendments against M. javanica. Some of the phytochemicals contained in cavalcade leaves, including flavonoids, phenols and terpenoids, were also quantified.
Materials and methods
Nematode preparation
The RKN population used in the study was collected from infected tomato plants at a Juja farm in Kiambu country, Kenya (1°09′37.1″S, 37°04′13.5″E). Egg masses on the tomato plants were removed manually and inoculated onto 10-day-old okra seedlings grown in pots filled with 1:1 (w/w) sterilised soil and sand to multiply and maintain the nematode inoculum. To extract eggs and J2, 2-month-old infected okra roots were cut into small pieces (0.5-1.0 cm long), placed into 50 ml Falcon tubes filled with 30 ml of 0.6% NaOCl and shaken by hand for 2 min 30 s (Meyer et al., 2020; Beesa et al., 2022). The nematode suspension was poured through nested 90 and 25 μm aperture sieves and rinsed under running tap water until clean. The J2 and eggs, collected on a 25 μm aperture sieve, were incubated at 27°C for 7 days for use as inoculum.
Nematode identification
Morphological identification by perineal pattern
Thirty adult RKN females were removed from the cultured okra roots for use to identify to species by perineal pattern analysis. Briefly, one adult female was transferred to a drop of water and cut using a razor blade to separate the anterior and posterior parts on a glass slide under a stereomicroscope (HSZ-ZB700, Huvitz). The perineal areas were transferred to a drop of 45% lactic acid for trimming and cleaning and then to a drop of distilled water mounted on a glass slide, covered with a coverslip, and sealed with nail polish. The slide was labelled and placed under a compound microscope (Leica DM750) for morphological observation according to Hartman & Sasser (1985).
Molecular identification
For molecular identification, seven adult RKN females were extracted from infected okra plants. The DNA was extracted using the method of Holterman et al. (2006) with slight modifications. In brief, one adult female was placed in a 0.5 ml PCR tube filled with 20 μl distilled water. Then, 20 μl lysis buffer (200 mM NaCl, 200 mM Tris-HCl pH 8.0, 1% (v/v) β-mercaptoethanol (all Sigma-Aldrich), and 800 μg ml−1 proteinase K (Meridian Bioscience)) was added and the mixture incubated at 65°C for 90 min, followed by 7 min at 99°C in a PCR Thermocycler (ProFlex, PCR System). The extracted DNA from each sample was stored at –20°C until use.
The PCR was performed using three primer sets: JB3/JB4.5 (5′-TTTTTTGGGCATCCTGAGGTTTAT-3′/5′-TAAAGAAAGAACATAATGAAAATG-3′) for cytochrome c oxidase subunit 1 (COI) mitochondrial gene (Bowles et al., 1992; Derycke et al., 2010), D2A/D3B (5′-ACAAGTACCGTGAGGGAAAGTTG-3′/5′-TCGGAAGGAACCAGCTACTA-3′) for partial 28S rRNA gene (Nunn, 1992) and Fjav/Rjav (5′-GGTGCGCGATTGAACTGAGC-3′/5′-CAGGCCCTTCAGTGGAACTATAC-3′) for species-specific primer (Zijlstra et al., 2000). A 20 μl PCR reaction included 3 μl of DNA template, 12 μl of sterilised distilled water, 0.5 μl each of 10 μM forward and reverse primers and 4 μl of 5×HOT FIREPol Blend Master Mix (Solis BioDyne). The PCR condition was performed as follows: D2A/D3B; denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min, and final extension at 72°C for 5 min, JB3/JB4.5; 94°C for 7 min, 5 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 30 s and temperature decreasing with 1°C for each cycle, and extension at 72°C for 30 s, followed by 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s, and a final extension of 10 min at 72°C and Fjav/Rjav; denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 64°C for 30 s and 72°C for 1 min, and final extension at 72°C for 7 min. Four representative samples of PCR products for each primer set were purified by rSAP and Exo I (BioLabs) and then sent to Macrogen Inc, Korea, for DNA sequencing. The obtained nucleotide sequences were aligned using ClustalW. Then, the aligned sequences were compared and deposited in the NCBI GenBank database.
Plant material and aqueous extract preparation
Cavalcade plants were grown in 25-cm-diam. pots filled with 1 kg sterilised paddy soil and maintained in a glasshouse, Faculty of Agriculture, Kasetsart University, at 25-33°C. Fertiliser (N-P-K, 16-16-16) was applied twice at 3 and 6 weeks after transplanting (3 g pot−1). Subsequently, 1-, 2- and 3-month-old cavalcade leaves were harvested, rinsed under running tap water and air-dried in the shade for 14 days. The dried plant materials were ground to fine particles in a blender (DXM-1000, DXFILL machine) and kept at room temperature until use.
To prepare aqueous extracts, 10 g of each ground material was added to 100 ml of distilled water in a 250 ml conical flask and shaken on an incubator shaker series (Innova44; New Brunswick Scientific) at 100 rpm for 24 h at 25°C. The mixtures were filtered through muslin cloths, centrifuged at 3000 g for 10 min, and the supernatants filtered through 0.45 μm and 0.22 μm syringe filters (Cole-Parmer) (Meyer et al., 2006). The obtained extracts were held at 4°C until use as stock solution (100% concentration) in microwell assays.
Second-stage juveniles hatching and mortality tests
Second-stage juvenile hatching and mortality assays with cavalcade leaf aqueous extracts were conducted in 96-well polystyrene plates, according to the procedures of Jindapunnapat et al. (2018). A 15 μl nematode suspension containing approximately 30 ± 5 eggs or J2 was added to each well in 185 μl of each diluted aqueous extract (25, 50 and 90% at final concentration). Streptomycin sulphate (Sigma-Aldrich) was used to prevent bacterial contamination. To prepare diluted aqueous extracts (185 μl), a stock solution (100%) was diluted by distilled water to give three concentrations: i) 25% = 50 μl of stock solution + 133 μl of distilled water + 2 μl of 50 mg ml−1 streptomycin sulphate; ii) 50% = 100 μl of stock solution + 83 μl of distilled water + 2 μl of 50 mg ml−1 streptomycin sulphate; and iii) 90% = 180 μl of stock solution + 3 μl of distilled water + 2 μl of 50 mg ml−1 streptomycin sulphate. Distilled water (0%) with 0.5 mg ml−1 streptomycin sulphate served as controls. The plates were covered with cling film and incubated at 27°C. The experiment included two factors and was arranged in a randomised completely block design (RCBD) with five replications and repeated once. The two factors were: i) age of cavalcade leaves; and ii) rate of application. The study included eleven treatments: 1) distilled water; 2) 0.5 mg ml−1 streptomycin sulphate; 3) 25% of 1-month-old cavalcade leaves (1MAE); 4) 50% of 1MAE; 5) 90% of 1MAE; 6) 25% of 2-month-old cavalcade leaves (2MAE); 7) 50% of 2MAE; 8) 90% of 2MAE; 9) 25% of 3-month-old cavalcade leaves (3MAE); 10) 50% of 3MAE; and 11) 90% of 3MAE. For hatching tests, the number of J2 that hatched was recorded at 7 days after exposure. For J2 mortality, inactive J2 were counted, after being rinsed three times with distilled water, at 2 days after immersion. At 72 h after incubation (3 days), dead J2 were re-checked. If the nematodes were not active they were considered dead. The percentage inhibition of hatching (Talavera-Rubia et al., 2020) and the percentage J2 mortality was calculated.
Glasshouse experiments
Preventive treatments
This study was conducted in the glasshouse at icipe campus located in Nairobi, Kenya, between July and September 2022. The daily temperature ranged between 11 and 28°C (AccuWeather, 2022). The powder of 1-, 2- and 3-month-old cavalcade leaves was mixed with 200 g of 1:1 (w/w) sterilised sand and soil (air-dried soil) with 0, 0.2, 0.5 and 1% (w/w) and filled in 7 cm diam. plastic pots. The mixtures were watered daily with 20 ml of tap water and incubated in the glasshouse for 6 days. Ten-day-old okra seedlings were rinsed free of soil under the tap and transplanted singly into each pot. Five days later, 500 eggs + J2 were inoculated in 1.5 ml water suspension into three 1.5 cm deep holes made around each plant using a pencil. Fertiliser (N-P-K, 16-16-16) was applied once at 7 days after nematode inoculation (0.1 g pot−1). The experiment included two factors and was arranged in a RCBD with four replications and repeated once. The two factors were: i) age of cavalcade leaves; and ii) rate of application. Data for fresh root weights, number of galls and number of egg masses (staining with phloxine B) were recorded at 5 weeks and 6 weeks after nematode inoculation for the two trials, respectively.
Eradicative treatments
This experiment was conducted using the same treatments and conditions as above, but the nematodes were inoculated into amended soil 5 days before transplanting okra seedlings. The fertiliser (N-P-K, 16-16-16) was applied once at 7 days after okra transplantation (0.1 g pot−1). Plants were harvested and data on root fresh weights, number of galls and egg masses were determined as for the preventive treatment experiment.
Quantification of phytochemicals
Aqueous crude extracts of 1-, 2- and 3-month-old cavalcade leaves were prepared as above. The crude extracts were freeze-dried for 5 days using a Wizard 2.0 freeze-dryer (SP Scientific). Each cavalcade leaf age was replicated six times. The obtained crude extracts were weighed and kept at 4°C until use.
Total flavonoid (TFC), phenol (TPC) and terpenoid (TTC) contents were quantified according to Kegode et al. (2022). A 0.25 g aqueous crude extract were each dissolved in 2.5 ml of 50% methanol. The obtained crude extracts were used to determine the following phytochemical contents. Each analysis of phytochemicals was carried out in triplicate.
Flavonoid content was determined using the aluminium chloride (AlCl3) colorimetric assay described by Popova et al. (2005). Briefly, 1 ml of obtained extract was mixed with 4 ml of distilled water and 300 μl of 5% (w/v) NaNO2 and incubated for 5 min. Then 300 μl of 10% AlCl3 was added and left for 1 min prior to adding 2 ml of 1 M NaOH and 2.4 ml of distilled water at the final step. The mixtures were centrifuged at 1300 g for 2 min. The total flavonoid content was determined by measuring the absorbance at 510 nm using a UV spectrophotometer (BioSpec). Quercetin (QE) at different concentrations (20-200 μg ml−1) served as a standard (
Phenol content was established using the Folin–Ciocalteu method (Popova et al., 2005). In brief, 5 ml of 0.2 M Folin-Ciocalteu reagent was added to 1 ml of extract and left at room temperature for 5 min before adding 4 ml of 75 g l−1 Na2CO3 and leaving for a further 1 h. The mixtures were then centrifuged at 1300 g for 2 min. The absorbance of the mixture was measured at 760 nm in a UV spectrophotometer. Gallic acid (GAE) at different concentrations (0-250 μg ml−1) served as a standard (
Terpenoid content was determined using the colorimetric method (Malik et al., 2017) by adding 1.5 ml chloroform to 200 μl crude extract, and thoroughly mixing using vortex for 1 min. Then 100 μl of concentrated H2SO4 was added and incubated in the dark for 1 h at room temperature. The supernatant was gently removed, leaving the reddish-brown precipitate at the bottom and 1.5 ml of absolute methanol added and mixed using vortex to dissolve the precipitate. The mixtures were centrifuged at 1300 g for 2 min and the terpenoid content estimated from the standard curve plotted using 0-500 mg ml−1 linalool (LE) (
Phytotoxicity assessment on okra
The mean GI values were compared to phototoxicity scores (Barral & Paradelo, 2011) as follows: GI < 50% represents high phytotoxicity; GI 50-80% represents moderate phytotoxicity; GI > 80% represents no phytotoxicity; and GI > 100% represents capability to be a phytonutrient.
Statistical treatment of data
All data were presented as means ± standard error. The data were statistically analysed using the SPSS software (version 16.0; SPSS). Differences among means were determined by Tukey’s multiple comparison (
Results
Nematode identification
The perineal patterns of all RKN females assessed were round or oval-shaped, with moderately high dorsal arch and obvious double lateral lines, which matched with the descriptions of M. javanica (Hunt & Handoo, 2009). The DNA amplification resulted in a specific amplicon size of 400, 750 and 670 bp for the three primer sets, which gave a 98-99% similarity to M. javanica sequences available in GenBank, confirming the identity as M. javanica. All sequences were deposited in GenBank under the accession numbers OP646643-OP646646 for COI, OP802558- OP802561 for D2-D3 and OP972598-OP972601 for the species-specific primers.
Effect of aqueous extracts of cavalcade leaves at different ages (factor A) and dosages (factor B) on percentage inhibition of Meloidogyne javanica hatch. Bars refer to standard error of the mean from two trials (n = 10). Similar lower-case letters in each value indicated that means are not significantly different according to Tukey’s multiple comparison at 0.05 level. **Significant at the 0.01 level.
Citation: Nematology 25, 9 (2023) ; 10.1163/15685411-bja10273
Hatching test
The effect of leaf age and dosage on hatching of M. javanica was analysed using a two-way ANOVA. The 50% and 90% concentrations of all cavalcade leaf ages were effective at reducing the hatch of M. javanica after immersion for 7 days (Fig. 1). By contrast, the 25% concentration of all leaf ages did not inhibit hatch when compared with the water and 0.5 mg ml−1 streptomycin sulphate controls. Maximum hatch inhibition (82.9%) was observed with a 90% concentration derived from 1MAE, followed by 90% 2MAE (49.9%) and 50% 1MAE (37.5%). At the 50% concentration, hatch was similar among different leaf ages. However, at the 90% concentration, younger leaf age (1MAE) showed higher potency in inhibiting hatch, as compared to the older leaf ages (2MAE and 3MAE).
Juvenile mortality test
The two-way ANOVA indicated that there was an interaction between the age of leaves and the dosage on percentage J2 mortality at 72 h after immersion. The 25% concentration for any leaf age had no adverse effects on J2. However, the 50% and 90% concentrations derived from 1-month-old leaves (1MAE) caused markedly greater J2 mortality than the same concentrations from 2- and 3-month-old leaves (2MAE and 3MAE). At 48 h after immersion, inactivity of J2 reached 88.5-100% for all concentrations across all leaf ages, compared with the water (1.2%) and streptomycin sulphate (3.4%) controls (Table 1). However, at 72 h, over half of the J2 resuscitated from their inactive state, across treatments. The greatest inhibitory effects on J2 (85% mortality) were recorded for 90% 1MAE followed by 90% 2MAE (55.8%), 90% 3MAE (52.7%) and 50% 1MAE (47.4%).
Percentage inactivity and mortality of Meloidogyne javanica infective second-stage juveniles (J2) after exposure to different concentrations and ages of cavalcade leaves.
Citation: Nematology 25, 9 (2023) ; 10.1163/15685411-bja10273
Effect of dried cavalcade leaf tissue of three different ages (1, 2 and 3 months) incorporated into soil at three rates on okra fresh root weights, number of galls and egg masses of the root-knot nematode (RKN) Meloidogyne javanica in a glasshouse in Kenya.
Citation: Nematology 25, 9 (2023) ; 10.1163/15685411-bja10273
Glasshouse experiments
No phytotoxic effects were observed in either of the two experiments (preventive or eradicative) (Table 2; Fig. 2). In addition, interactions were observed in amended rates (factor B) on suppressing M. javanica in both preventive and eradicative treatments. The 0.5% and 1% amended rates of cavalcade leaves considerably reduced the number of galls (g root)−1 and egg masses (g root)−1. RKN suppression increased with increasing rate of cavalcade leaf concentration. The 1% 1-month-old (both preventive and eradicative treatments) leaves was the most effective at reducing M. javanica damage.
Quantification of phytochemicals
The weight of aqueous crude extracts derived from 1 g dried cavalcade leaves revealed that crude extracts were similar between the three different leaf ages, although there was a relatively higher quantity in 1-month-old (98.89 mg) leaves. The information obtained reflects the tested concentrations of the above experiments as follows: bioassays with 90% of 1MAE, 2MAE and 3MAE were 89, 86 and 80 mg ml−1, respectively, and soil amendments with 1% of 1-, 2- and 3-month-old leaves were 0.99, 0.96 and 0.89 mg (g soil)−1, respectively.
Of the three phytochemical groups, higher concentrations of flavonoids (26.8 mg QE g−1) and phenols (2.35 mg GAE g−1) were detected in aqueous cavalcade leaf crude extracts from 1-month-old leaves (Fig. 3). However, the phenol content was not significantly different from 3-month-old leaves (2.25 GAE g−1), while terpenoid contents were lower in 1-month-old (90.4 mg LE g−1) than 2- (133 mg LE g−1) and 3-month-old (131.4 mg LE g−1) leaves.
Okra roots grown in soil amended with 1% cavalcade leaves and inoculated with the root-knot nematode (RKN) Meloidogyne javanica. A: Preventive treatments: seedlings inoculated 5 days after transplanting; B: Eradicative treatments: seedlings inoculated 5 days before transplanting.
Citation: Nematology 25, 9 (2023) ; 10.1163/15685411-bja10273
Quantification of flavonoid (A), phenol (B) and terpenoid (C) contents derived from aqueous crude extracts of 1-, 2- and 3-month-old cavalcade leaves. Bars refer to standard error of the mean (n = 3). Similar lower-case letters in each figure indicated that means are not significantly different according to Tukey’s multiple comparison at 0.05 level. QE = Quercetin Equivalent, GAE = Gallic Acid Equivalent, LE = Linalool Equivalent.
Citation: Nematology 25, 9 (2023) ; 10.1163/15685411-bja10273
Phytotoxicity assessment on okra
The radicle length of okra seedlings and the percentage seed germination were unaffected following soil amendment with dried cavalcade leaves (Table 3). No adverse effects on okra seedlings were observed with GI values ranging from 81.2 to 104.6%, portraying no phytotoxicity. Moreover, soil amendment with 3-month-old cavalcade leaves appeared to act as a phytonutrient. No interaction between the age of cavalcade leaves and rate of application was observed to influence the percentage seed germination. Therefore, any leaf ages or amended rate uses as soil amendments produced the same results, which did not pose negative effects to seed germination.
Discussion
This current study demonstrates the potential for the leguminous crop C. pascuorum to contribute to the management of RKN. Although experiments were mostly in vitro, results clearly showed effective inhibition of hatch and J2 mortality of M. javanica, leading to reduced root galling damage and egg mass production under glasshouse conditions. Our study further revealed that fresher or younger leaf age appeared to be more potent with higher inhibitory effects on the nematode. This may be due to the loss of nematicidal compounds, such as flavonoids, phenols and terpenoids, with age of the C. pascuorum leaf extracts. Such volatile compounds are known to play an important role against M. javanica (Chitwood, 1992). The higher concentrations of the extracts also corresponded with higher nematode suppression, while no negative phytotoxicity effects were observed at the application rates used in this study. Our study reflects results from a separate study, using the leguminous cover crops C. pascuorum and S. guianensis that suppressed Hirschmanniella spp. population densities in rice fields (Suong et al., 2019; Beesa et al., 2021). However, to date no reports have described the governing mechanism of C. pascuorum for controlling the nematodes.
The highest concentration (90%) 1 MAE of the aqueous extracts of cavalcade leaves showed the greatest inhibitory effects on M. javanica and the aqueous extracts derived from 1-month-old cavalcade leaves displayed more effective reduction of M. javanica than those obtained from 2- and 3-month-old leaves, indicating that there is a loss of potency with age. Similar results were reported by Jindapunnapat et al. (2018), who noted that aqueous root and shoot extracts derived from younger plants (2 months old) of V. zizanioides showed greater activity against M. incognita J2 than older plants (4 years old). Other studies have also demonstrated that aqueous extracts of leguminous leaf material control RKN, such as Mucuna pruriens, Crotalaria spectabilis and Dolichos lablab (Jourand et al., 2004; Osei et al., 2010). Aqueous extracts from Cajanus cajan, Crotalaria and C. cajan + Crotalaria also led to 100% nematicidal effect on Scutellonema bradys (Garrido et al., 2008), while extracts from a range of legume crops have conversely been shown to possess no nematicidal activity, including water extract of Crotalaria seeds (Harender et al., 2012). With regard to the use of cavalcade aqueous leaf extracts, the current study is the first such study to assess and document its potential for nematicidal activity against RKN, specifically M. javanica.
The use of cavalcade leaves as a soil amendment to protect plants against RKN was also demonstrated in the current study. The use of the youngest (1-month-old) leaves showed greatest impact on M. javanica infection of okra in the glasshouse. Although all treatments reduced the number of galls and egg masses on roots, the 1% amended rate of 1-month-old leaves were most effective. This result reflects studies by Kankam et al. (2015) and Patel & Dhillon (2017) using sunn hemp (C. juncea) as a soil amendment, which significantly decreased M. incognita densities by 51-94%. Another study found that incorporation of 10% fresh vetiver into the soil significantly reduced M. incognita by 46-67% (Jindapunnapat et al., 2019). The use of cavalcade and S. guianensis (‘Nina’) as cover crops in rice fields in Cambodia has been associated with reduced densities of M. graminicola and Hirschmaniella sp. (Beesa et al., 2021; Masson et al., 2022). Our current study confirms the suppressive effect of cavalcade and indicates that it is likely due to nematotoxic compounds. In terms of the effect of cavalcade leaf amendment on seed germination, no negative impact was observed on okra seed germination. Moreover, soil amended with 3-month-old cavalcade leaves appears to act as a phytonutrient. Similar results have been reported by Moldes et al. (2007), who recorded GI value > 125 (act as phytonutrient) of rye grass in composted grape pomace. Islam & Faruq (2008) also found that soil amended with neem compost significantly increased tomato seed germination (29.85%), root length (48.22%) and shoot length (72.25%). When using S. guianensis and C. pascuorum as cover crops, soil fertility (soil organic matter) and rice yields in Cambodian were shown to increase (Suong et al., 2019; Beesa et al., 2021).
In our study, the greatest nematicidal activity was observed with the youngest leaf material, which may be associated with higher bioactive compounds in crude extracts of flavonoids and phenols. The presence of flavonoid, phenol and terpenoid phytochemicals are likely associated with nematicidal activity, as indicated in other studies (Chitwood, 1992; Chen & Song, 2021). Several flavonoid compounds, e.g., flavonols, kaempferol, quercetin, and myricetin, are also known to repel RKN J2 and to inhibit hatch (Wuyts et al., 2006), due to slowing the movement of J2, modifying nematode orientation to the plant roots and killing nematodes (Chin et al., 2018). From 49 phenolic compounds screened against M. incognita, d-(−)-4-hydroxyphenylglycine, t-butylhydroquinone, l-3-(3,4-dihydroxyphenyl) alanine, sesamol, 2,4-dihydroxyacetophenone, and p-anisaldehyde were found to be lethal to RKN J2 (Oliveira et al., 2019), whilst various studies have shown the nematicidal effect of a range of phenolic compounds (Ohri & Pannu, 2010) and other volatiles (Abdel-Rahman et al., 2013). Echeverrigaray et al. (2010) similarly showed significant M. incognita hatch reduction by monoterpenoids and reduced J2 mobility at a concentration of 0.25 mg ml−1. Therefore, this study reveals that the nematicidal activity of cavalcade leaves on M. javanica could be associated with the presence of flavonoid, phenolic and terpenoid compounds. However, additional studies are needed to clarify the nematicidal compounds present in cavalcade leaves that are responsible for controlling M. javanica.
Influence of cavalcade leaf age and concentration on okra seed germination under glasshouse conditions in Kenya.
Citation: Nematology 25, 9 (2023) ; 10.1163/15685411-bja10273
In conclusion, our study provides strong evidence on the nematicidal effects of cavalcade and its potential as a bio-based option for nematode management. In the current study, it was demonstrated that aqueous extracts of dried leaves achieved high efficiency in controlling M. javanica in both in vitro and glasshouse settings. The controlling mechanism may be associated with the existence of nematicidal flavonoid, phenol and terpenoid volatile compounds that appear to reduce in potency with age of the leaves. More extensive research is naturally required to determine further the real potential of cavalcade for nematode management on a field basis. However, this study provides a sound and promising basis upon which to build.
Corresponding authors, e-mails: agrbcc@ku.ac.th; d.coyne@cgiar.org
Acknowledgements
The authors extend their gratitude to Molecular Biology and Bioinformatics, Arthropod Pathology, and Behavioural and Chemical Ecology Units, icipe, for equipment support. We also acknowledge Mr Hosea O. Mokaya, Mr Timothy M. Kegode and Prof. Dr Baldwyn Torto for their guidance on phytochemical analyses. We would also like to thank NemAfrica team and Kaset NemLab members for their help. This work was financially supported by the Office of the Ministry of Higher Education, Science, Research, and Innovation and the Thailand Science Research and Innovation through the Kasetsart University Reinventing University Program 2021.
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