The trial was conducted in autumn (March-May) 2019 and repeated in 2020 under field conditions at the Green Biotechnologies Research Centre of Excellence, University of Limpopo (23°53′10″S, 29°44′15″E), South Africa. The location is semi-arid with summer rainfall averaging less than 450 mm per annum and summer temperature averaging 28^°C. Hutton soil at the experimental site comprised 65% sand, 30% clay, 5% silt, which was thus classified as loam, with pH (H₂O) 6.5 and EC 1.87 dS/m.

Nemafric-BL phytonematicide was prepared through fermentation using oven-dried fruit from cultivated wild watermelon (Cucumis africanus L.) and effective microorganisms (EM) as described previously (Mashela et al., 2017a). Biocult and Nemafric-BL phytonematicide, as the first and the second main factors, respectively, were laid out in a 2 × 2 factorial experiment. The four treatments (B₀P₀, B₁P₀, B₀P₁, B₁P₁) were arranged in randomized complete block design with 10 replications (n = 40), with blocking being for shading by windbreaks in the morning and in the afternoon. Due to the factorial nature of the experiment, there was no need to repeat the experiment (Little and Hills, 1978).

A drip irrigation line was laid out resulting in planting stations at 1.0 m × 0.45 m spacing, with each drip hole discharging 2 L chlorine-free water/h. A day before sowing, each station was irrigated with 3 L half-strength Hoagland solution without N (Hoagland and Arnon, 1950). Primed cowpea var. Eureka seeds were inoculated with Rhizobium leguminosarium biovar phaseoli spores while still moist and placed on laboratory bench for 3 days prior to sowing. Four seeds were sown per drip hole and at 5 days after complete emergence, seedlings were thinned to one per drip hole.

Soil samples were collected adjacent to each seedling using a Soil Sampler Probe 52 cm (Mylawncare, Johannesburg, South Africa), composited and mixed. Second-stage juveniles (J2) were extracted from 250 ml soil subsample using the modified sugar-centrifugation floatation method (Marais et al., 2017), with J2 counted under a 60 × magnification stereomicroscope, with mounted specimen identified as M. enterolobii using morphometric characters. The initial nematode population density (Pi) averaged 35 J2/250 ml soil subsample, which necessitated augmentation of each seedling with 250 eggs + J2 M. enterolobii collected from roots of tomato plant cv. “Floradade.” Eggs + J2 were dispensed using a 20 ml plastic pipette around each seedling with holes covered using soil. Five days after thinning, Biocult was applied by placing 2 g in a shallow furrow around each seedling and covering with soil. Five days thereafter, Nemafric-BL phytonematicide was applied at 2%, with biweekly reapplication until harvest. Aphid populations were monitored daily, with plants sprayed once at 35 days after seedling emergence using malasol at 5 ml/5 L chlorine-free water to contain the aphid infestations.

At 60 days after the phytonematicide treatment, whole plants were removed. Plant length was measured from the severed end to the tip of the flag leaf of the longest vine using a measuring tape. Stem diameter was measured at 5 cm above the severed distal end using a digital Vernier caliper. Tender leaves usually harvested for use as leafy vegetable were harvested and dried with shoots in air-forced ovens at 60°C for 72 h for dry leaf and shoot mass. Root systems were removed from each station, immersed in water to remove soil particles, blotted dry, with eggs and J2 extracted from 10 g roots using the modified sugar floatation method (Marais et al., 2017). From each planting site, 300 ml soil sample was collected, mixed and J2 extracted from a 250 ml soil subsample (Marais et al., 2017), with root and soil nematodes separately counted under a stereomicroscope. Nematode data were transformed using log₁₀(x + 1) to normalize the variances. All datasets were separately subjected to the Shapiro-Wilk test using Statistix 10 software to assess the normality of the distribution of data (Shapiro and Wilk, 1965; Ghasemi and Zahedias, 2012). The data exhibited normal distribution and were therefore subjected to factorial analysis of variance.

At harvest, the interaction of Biocult and Nemafric-BL phytonematicide was highly significant (P ≤ 0.01) on plant growth variables. Relative to untreated control, Biocult alone increased plant variables from 15 to 74% (Table 1). Similarly, NemafricBL phytonematicide increased plant variables from 14 to 61%. However, the combined effects significantly increased dry shoot mass by 19% and dry harvestable leaf mass by 21%, but did not have significant effects on plant height and stem diameter.

The interaction of the two products was also highly significant on nematode variables Relative to untreated control, Biocult alone reduced nematode eggs in root, J2 in root and J2 in soil by 80, 84, and 53%, respectively (Table 2). However, the combined effects of the two products did not have significant effects on any of the test nematode variables in roots, but significantly reduced J2 in soil by 30%.

In the current study Biocult and Nemafric-BL phytonematicide each significantly increased growth variables of cowpea plants. Glomus species and Trichoderma species in Biocult have the potential of increasing soil biological activities that improve soil health by inducing the root system to exudate various secondary metabolites, including enzymes, into the rhizosphere (Stirling, 2014; Yan et al., 2021). Additionally, Glomus species, the arbuscular mycorrhizal fungi, provide benefits that include improved absorption of phosphorus and certain micronutrient elements (Richardson et al., 2011) and nematode suppression (Gough et al., 2020). The species can also accord the host plant the ability to access water beyond the normal wilting point, thereby according the plants the drought tolerance status, particularly under suboptimal conditions (Proveda et al., 2020).

Another endophytic fungi, Trichoderma species, in addition to promoting plant growth through various mechanisms (Ozbay and Newman, 2004), which include mineral solubilization and boost nutrient uptake, also provide certain degree of protection against soil pathogens, including nematodes (Stewart and Hill, 2014). Colonization of roots by Trichoderma species had been associated with improved chlorophyll content, alleviation of abiotic stress, increased exudation of secondary metabolites and the production of various plant growth regulators hormones (Yan et al., 2021). However, the effects of Glomus species and Trichoderma species on plant growth are plant- and species-specific (Stewart and Hill, 2014; Gough et al., 2020). Combined, Glomus species and T. asperellum in Biocult were previously observed to have synergistic effects on increasing dry shoot mass in cucumber (Cucumis sativa L.) plants when compared with individual treatment effects (Chandanie et al., 2009).

In our study, Nemafric-BL phytonematicide had consistent effects on improving growth variables of cowpea plants. In other studies, where the product was used to manage nematode population densities of M. incognita and M. javanica, the product consistently improved crop growth variables (Mashela et al., 2017a). Cucurbitacins in phytonematicides can either have stimulation, neutral or inhibition effects on plant growth. However, since the dosage accumulates in the rhizosphere with repeated phytonematicide application, response in different plant organs can also be concentration-specific as shown in different studies (Mashela et al., 2017a). Cucurbitacin B in Nemafric-BL phytonematicide is able to penetrate the apoplastic pathways in roots, but cannot penetrate the symplastic pathway which is accorded by the pericycle and the endodermis in root systems (Van Wyk and Wink, 2014). In our study, as shown by the plant growth responses, concentration of cucurbitacin B in apoplastic spaces remained within the stimulation range for all the test plant variables since growth was significantly increased.

Notably, the combined effects of Biocult and Nemafric-BL phytonematicide, had antagonistic effects on plant growth when compared with that of either of product. In colonized root systems, mycelia of the filamentous fungi penetrate the root system to establish mutual relationships (Stewart and Hill, 2014; Proveda et al., 2020). Such relationships change the morphology and metabolism of the root system, along with the bioactive chemical compounds of affected plant roots (Vasundhara et al., 2016; Yan et al., 2021). Apparently, when the two products are combined, there are both physical and chemical limitations in roots. Firstly, the proliferation of mycelia in the apoplast spaces, alter the available physical spaces in which free water that carries cucurbitacin molecules perculate. Consequently, the reduced free-passage of free water, invariably results in reduced concentration of cucurbitacin B in apoplastic spaces. Secondly, although reduced cucurbitacin B could have stimulation effects on plant growth variables, for lower organisms such as Glomus and Trichoderma species, the concentration could have been at the inhibition range, technically, the cytotoxic range. Under in vitro conditions, which mimic apoplastic conditions, T. harzianum suppressed mycelia of G. intraradices (Green et al., 1999).

Cowpea var. Eureka is a host to Meloidogyne species (Mashela and Pofu, 2012). In our study, Biocult and Nemafric-BL phytonematicide each consistently suppressed nematode population densities of M. enterolobii in root and in soil, which confirmed observations in other studies (Mashela et al., 2017a; Proveda et al., 2020; Yan et al., 2021). Trichoderma species are primarily biocontrol agents that have inherent capabilities of triggering plants to release chemicals with nematicidal properties (Proveda et al., 2020; Yan et al., 2021). Such chemicals include flavonoids, phenols and chitinase, β-1,3 glucanase, protease, amylase, which are known as plant genes that confer nematode resistance in plants (Mashela et al., 2017b). The induced plant resistance as observed in var. Eureka in our study, had been referred to as systemic acquired resistance (Stirling, 2014; Proveda et al., 2020). The effects of Nemafric-BL phytonematicide on population densities of M. enterolobii in the current study, confirmed those of this product on a wide range of other Meloidogyne species and races (Mashela et al., 2017b), which we do not wish to discuss further in the current study.

Both Glomus species and Trichoderma species are endophytes, with different roles and capabilities of inducing nematode resistance in plants (Proveda et al., 2020). However, the two species, when combined, were at times shown to have significant antagonistic effects on population densities of Meloidogyne species (Yan et al., 2021), with the effects not being limited to the induced chemicals. After J2 hatch, J2 move out of roots into soil solution, from where search for penetration sites occurs. After penetration, J2 move to the tip of root and enter the vascular bundle and then move upward through the xylem to the zone of differentiation, where feeding sites are established (Mashela et al., 2017b). In addition to inducing chemicals with nematicidal properties (Yan et al., 2021), mycelia and spores of the endophytes can physically impede J2 mobility (Stewart and Hill, 2014; Gough et al., 2020; Proveda et al., 2020).

Generally, the effects of cucurbitacins on entities are both concentration- and size-specific. Concentrations that have stimulation effects on plant growth were previously shown to have significant inhibition effects on various bioactivities on minute entities like plant nematodes (Dube, 2016). In Nemarioc-AL phytonematicide, with active ingredient cucurbitacin A, which is partly polar and therefore slightly soluble in water. Nodules induced by Bradyrhizobium japonicum on cowpea vs. increasing concentration of Nemarioc-AL phytonematicide exhibited quadratic positive relationships, with nodules being highly sensitive to the product (Kola et al., 2018). Although in the current study we observed consistent antagonistic effects of Biocult and Nemafric-BL phytonematicide, T. harzianum in short-term studies was tolerant to Nemarioc-AL and Nemafric-BL phytonematicides at concentrations used in nematode management (Madaure et al., 2019). In our study, Nemafric-BL phytonematicide was applied biweekly, with the likelihood that the stable cucurbitacin B accumulated within the apoplastic spaces, thereby inhibiting both mycelia and spore development. In other studies (Vasundhara et al., 2016; Proveda et al., 2020) the combined effects of Trichoderma species with either Paecilomyces lilacinus biocontrol agent, neem (Azadirachta indica A. Juss) phytonematicide or castor (Ricinus communis L.) oil had synergistic effects in suppressing population densities of Meloidogyne species, which contradicted observations in our study. However, our findings confirm the assertion that the efficacy of filamentous fungi on inducing beneficial effects is dependent upon the fungal species, plant species and pathogen type and species (Gough et al., 2020). Additionally, in interaction with agricultural remedies of botanical nature, the degree of sensitivity of filamentous fungi to the active ingredients of the test product could also play an indispensable role in the expression of the interactive effects.