Soil-borne microbial and invertebrate pests and diseases are a major problem to crop production around the world. Of particular importance are disease complexes involving plant-parasitic nematodes and numerous fungal pathogen strains that cause root rots and wilt diseases (Back et al., 2002; Zhang et al., 2020). In banana production, Radopholus similis is among a complex of plant-parasitic nematodes that interact with the Fusarium of Banana Complex (FOBC) in causing root rots and wilt diseases (Speijer and Sikora, 1993; Ochieno, 2020; Rocha et al., 2020). Control of such disease complexes has focused on developing pest and disease resistant banana varieties (Tripathi et al., 2017; Siamak and Zheng, 2018), and multiplying them as clean planting material through aseptic cell and tissue culture technologies (Almekinders et al., 2019; Jacobsen et al., 2019).

Aseptic tissue culture protocols target the elimination of complexes of non-beneficial endophytic and soil-derived biota that are associated with plant growth suppression (Cassells, 2012; Kumar et al., 2021). Endophytic and rhizosphere plant growth promoting microbes are re-introduced to restore their beneficial functions lost through aseptic tissue culture and soil sterilization techniques (Niere et al., 1999; Sikora and Pocasangre, 2006; Sikora et al., 2008; Soumare et al., 2021). For instance, Fusarium oxysporum strain V5w2 is among endophytic microbes that have successfully been re-introduced into tissue culture banana plants and transferred to farmers in East Africa (Dubois et al., 2006, 2011; zum Felde, 2011). Induced systemic resistance is among the key mechanisms said to be involved in pest suppression by F. oxysporum V5w2 among other microbial biological control agents (Vu et al., 2006; Paparu et al., 2007, 2009a,b, 2010; Bubici et al., 2019; Damodaran et al., 2020). However, future research is still necessary to elucidate the mechanisms underpinning nematode suppression and increased banana yield observed after treatment with F. oxysporum V5w2 (Kisaakye et al., 2022). This is partly because F. oxysporum V5w2 has been reported to be a non-beneficial endophyte that interacts with R. similis in a wilt disease complex of banana (Ochieno, 2020, 2021).

Assessments of the improved banana varieties for resistance to soil-borne pests and diseases, as well as the function of plant growth promoting microbes, have been primarily performed through controlled screenhouse experiments utilizing potted plants and sterilized soil substrates (Dubois et al., 2004; Paparu et al., 2009b; Waweru et al., 2013). This way, integrated pest management (IPM) strategies for banana are developed.

Experiments with potted plants have often produced varied results. For instance, infections by strains of Fusarium oxysporum have been associated with effects ranging from being positive, neutral, or negative toward the host banana plants and nematode pests (Athman et al., 2007; Paparu et al., 2009b; Ochieno, 2010, 2020). Studies that only feature potted banana plants and nematode pests in sterile soil fall short of information on the contribution of fungal pathogens toward parasitism (Hasan, 1993; Ochieno, 2010, 2020). This is also the case for studies that assess potted banana plants and fungal pathogens in sterile soil without plant-parasitic nematodes (Ochieno, 2010, 2020; Khan and Sharma, 2020). These two previous scenarios fail to address the natural interactions between plant-parasitic nematodes and fungal pathogens in root disease complexes. Moreover, possible roles of rhizosphere biota that exist in agroecosystems cannot be addressed through experiments that only feature sterilized soil (Ochieno, 2010, 2020; Kuijken et al., 2015; Jayaraman et al., 2021). Sterile soil may still be recolonized by different microbes (Li et al., 2019), while the axenic explants may still contain endophytic microbes or get re-infected (Ochieno, 2010, 2020; Thomas and Sekhar, 2017; Kithaku et al., 2019).

Abiotic factors especially nutrient compounds have been included into experiments to clarify their effects on interactions between plants, antagonistic and beneficial soil biota. This includes approaches that involve nutrient omission experimental treatments (Kolawole et al., 2018; Mugo et al., 2021). Where nutrient uptake by potted plants is not addressed, depletion of such resources could lead to variations in results. Comparison of plant-microbe-nematode interactions under different nutrient regimes produces varied results with different interpretations (Ochieno, 2010). Although differences in nutrient content and concentration are usually assumed, variations in other physico-chemical properties of the formulations including pH, salinity, conductivity, and toxicity may also affect the outcomes. Therefore, there is dilemma on how results from experiments utilizing nutrient formulations varying in multiple physico-chemical attributes should be interpreted (Ochieno, 2010). This is even further confounded by changes in soil physico-chemical composition that result from sterilization (De Deyn et al., 2004; Gundale et al., 2019; Dietrich et al., 2020). Experiments on plant-microbe-nematode interactions would therefore be better interpreted when conducted under a standard nutrient regime (Lancien et al., 1999; Shin et al., 2005; Ochieno, 2010), while addressing the contribution of rhizosphere microbes (De Rooij-van der Goes, 1995; Troelstra et al., 2001; Ochieno, 2010, 2020).

The present work is part of studies that are aimed at providing information on interactions between plant-parasitic nematodes and fungal pathogens, with specific focus on R. similis and F. oxysporum V5w2 in tissue culture banana (Ochieno, 2020). The studies explore how the two root parasites interact with soil biota under nitrogen (N), phosphorus (P), and potassium (K) deficient conditions. It is hypothesized that, pathogenic interactions between R. similis and F. oxysporum V5w2 under N, P, and K deficient conditions are enhanced by a complex of non-beneficial soil microbes that exert plant growth suppressive effects in tissue culture banana plants.

The present three experiments were conducted to find out whether, pathogenic interactions between R. similis and F. oxysporum V5w2 under nitrogen (N), phosphorus (P), and potassium (K) limited conditions, are enhanced by a complex of non-beneficial soil microbes that exert plant growth suppressive effects in tissue culture banana (Kumar et al., 2021). Plant parasitic activities of R. similis on banana plants were evident in the form of increased density of the nematode that resulted in root damage through necrosis, death and reduced plant biomass. This root burrowing nematode is known to cause such root damage that inhibits water and nutrient uptake from the rhizosphere resulting in poor growth and toppling of banana trees (Devi et al., 2009; Haegeman et al., 2010; Seenivasan, 2017). Root damage by R. similis also allows co-infections by root-invading organisms such as microbial pathogens and other plant-parasitic nematodes involved in plant disease complexes (Khan and Sharma, 2020). Such root-invading organisms may have included the 25 endophytic fungi that were isolated from the banana root pieces among them Fusarium spp., Rhizoctonia spp., Trichoderma spp., Sclerotium spp., Bipolaris spp., Curvularia spp., Mucor spp., Rhizopus spp., Papulaspora spp., Pleurophragmium spp., and Trichocladium spp.; as well as plant-parasitic nematodes that included Helicotylenchus multicintus, Meloidogyne spp., Pratylenchus goodeyi, Xiphinema spp., Trophorus spp., Scutellonema spp., Rotylenchus spp., Tylenchus spp., Hemicycliophora spp., Paratrichodorus spp., and Ditylenchus spp.

Contrary to expectations, N-starved banana plants inoculated with R. similis exhibited better growth in terms of shoot biomass, height, number, and size of leaves. The N-starved banana plants inoculated with R. similis even tended to have higher chlorophyll content that made them appear less yellow or otherwise greener. Probably, N in the form of ammonium excreted by R. similis feeding on the roots is preferentially directed toward chlorophyll synthesis under N-deficient conditions. This is because the nematode-excreted ammonium (NH4+; Neher, 2001; Zinov'eva et al., 2004), is the most readily available form of nitrogen for assimilation by plants (Mur et al., 2017; Ochieno et al., 2021). Also, root-feeding activities of R. similis may have enhanced the rhizodeposition of carbon-rich compounds including enzymes that favor the acquisition of scarce N from soil organic matter (SOM; Gebremikael et al., 2016). Rhizodeposition is part of physiological and morphological changes that include elongation of roots which enable N-starved plants to survive the adverse conditions (Bowsher et al., 2018).

Root infection by R. similis appeared to be beneficial to Fusarium spp. through creation of wounds and necrotic tissues for saprophytic growth of the fungus (Back et al., 2002; Khan and Sharma, 2020). This manifested in the form of enhanced root piece colonization under the three nutrient deficient regimes (Ochieno, 2010, 2020). F. oxysporum V5w2 was expected to cause wilt symptoms (Ochieno, 2020), which are related to pathogenicity of F. oxysporum races in susceptible banana cultivars (Dita et al., 2018; Maryani et al., 2019; Segura-Mena et al., 2021). However, there was no basis for linking F. oxysporum V5w2 with wilt-related pathogenicity in the N-starved banana plants. This is because the N-starved banana plants were already expressing nutrient stress symptoms including yellowing of leaves and stunted growth (Ochieno, 2010), which also occur in plants infected with pathogenic F. oxysporum races (Warman and Aitken, 2018). Possible pathogenic invasion of the N-deficient xylem may not have been nutritively beneficial to endophytic F. oxysporum (MacHardy and Beckman, 1981; Pegg et al., 2019), as necrotrophic and saprophytic colonization may have been a more viable nutritional strategy (Orr and Nelson, 2018; Pegg et al., 2019; Ochieno, 2020).

Inoculation of banana plants with F. oxysporum V5w2 is expected to suppress R. similis multiplication and the associated root damage in banana plants (Kumar and Dara, 2021; Kisaakye et al., 2022). This was the case in the study that utilized N-starved banana plants. Inoculation with F. oxysporum V5w2 under conditions of N-starvation was associated with reduced densities of R. similis and root damage by the nematode especially in the microbe-rich non-sterile soil. However, endophytic and rhizosphere colonization by F. oxysporum V5w2 may have intensified competition for the already limited N and hence inhibiting the multiplication of R. similis (Ochieno, 2020). Reduction in optical density of root extracts of banana plants infected with F. oxysporum V5w2 and R. similis may indicate competition for nutrient resources (Ochieno, 2010, 2020). Therefore, competition for N under its extreme deficiency (Ochieno, 2020), is an alternative to explanations that are based on the concept of induced systemic resistance (Vu et al., 2006; Paparu et al., 2007, 2010, 2013; Sikora et al., 2008; Bubici et al., 2019; Damodaran et al., 2020).

In all the three nutrient starvation regimes, banana plants grown in non-sterile soil had reduced densities of R. similis in roots, while root damage caused by the nematode was lower than in sterile soil. The synergistic effect of R. similis on root colonization by F. oxysporum V5w2 was suppressed in non-sterile soil in the presence of P within the N- and K-deficient nutrient solutions. These were indications of the presence of pest-suppressive microbes in non-sterile soil (Shen et al., 2015; Giné et al., 2016; Jayaraman et al., 2021). For instance, among the microbial genera isolated from banana roots in the present study, some that belong to Fusarium, Trichoderma, and Sclerotium have been associated with nematostatic activities (Akinsanmi and Adekunle, 2003; Poveda et al., 2020; Kumar and Dara, 2021). Furthermore, R. similis may have encountered antagonistic interactions with other nematode genera identified, especially through competition with plant-parasitic nematodes such as Helicotylenchus multicintus, Meloidogyne spp., and Pratylenchus goodeyi (Freckman and Caswell, 1985; Ochieno, 2010). Inclusion of F. oxysporum V5w2 in non-sterile soil even caused further reduction in root damage and R. similis densities among N-starved banana plants. Elimination of such antagonistic microbes through soil sterilization allowed R. similis to multiply and cause root damage. However, suppression of R. similis multiplication and the associated root damage in non-sterile soil did not improve the growth of N-starved banana plants. Instead, the N-starved banana plants grown in non-sterile soil were even smaller in terms of biomass and size. Increased demand and hence competition for N by soil microbes including F. oxysporum V5w2 in non-sterile soil (Jones et al., 2018), may have limited the growth of the N-starved banana plants and pest-related activities of R. similis. Such competition for N was manifested in the form of reduced chlorophyll content in leaves of banana plants grown in non-sterile soil. Inclusion of N in non-sterile soil reversed such trends toward suppression of root damaging activities of R. similis while improving banana plant growth, as was evident in the root biomass of plants that received P- and K-deficient solutions that contained adequate N. Therefore, microbial services cannot be well attained under conditions of poor soil fertility and plant nutrition, especially when N is limited. Beneficial soil microbes require proper nutrition to prevent them turning into competitors.

Plants inoculated with F. oxysporum V5w2 were smaller in size in terms of leaf development under K-starvation, with most plant growth related parameters inclining toward such trend under P-starvation. This is a pointer to the fact that the fungal pathogen F. oxysporum V5w2 may have initiated its plant growth suppressing effects within the short period (Ochieno, 2010, 2020). Under P-starvation, co-infection of banana roots with R. similis and F. oxysporum V5w2 resulted in increased density of the nematode and higher root colonization by Fusarium spp. Such trends in R. similis and Fusarium spp. densities appeared to manifest in roots of banana plants grown under K starvation. Densities of R. similis, Meloidogyne sp., and Pratylenchus sp. have also been found to increase in roots of F. oxysporum-inoculated field-grown banana plants (Ochieno, 2010, 2020, 2021). Such trends in R. similis and Fusarium spp. densities are indications of nematode-fungi disease complexes (Back et al., 2002; Khan and Sharma, 2020; Ochieno, 2020), which may be favored in soils that are deficient in P and K among other unfavorable ecological conditions (Almeida et al., 2018; Olivares et al., 2021).

Various speculations can been put forward to explain the positive outcomes of interactions between R. similis and F. oxysporum V5w2 under the P-limited conditions. First, it is worth noting that P is an important nutrient in root development (Crombez et al., 2019; Siedliska et al., 2021). Phosphorus is part of the phospholipids that form the plant cell membranes (Wang et al., 2017, 2021). P-deficiency is associated with weakened cell membranes (Gniazdowska et al., 1999; Pfaff et al., 2020), which may easily be penetrated by R. similis and F. oxysporum V5w2 resulting in increased colonization by the two pathogens. Phosphorus is a chemical component of adenosine triphosphate (ATP; Wang et al., 2021), which provides energy for biosynthesis of plant defensive compounds (Dhakshinamoorthy et al., 2014; Crombez et al., 2019; Desmedt et al., 2020). P-deficiency may limit the biosynthesis of such defensive compounds and render banana plants less resistant to R. similis and F. oxysporum V5w2. Under P-deficiency, F. oxysporum V5w2 may have released phosphatases to enhance acquisition of the limited nutrient from organic sources (Tarafdar et al., 2001; Nunez-Rodriguez et al., 2020). Such enzymes may have further degraded the weakened and damaged roots to facilitate penetration and colonization by R. similis and F. oxysporum V5w2. P-deficiency may also have triggered increased root exudation of organic carbon in the form of sugars into the rhizosphere (Louw-Gaume et al., 2010, 2017; Canarini et al., 2019). The organic carbon may have intensified rhizosphere colonization by the saprophytic F. oxysporum V5w2 (Ochieno, 2020; Clocchiatti et al., 2021), making root invasion, colonization and degradation even more severe for easier penetration and multiplication of R. similis. Potassium on the other hand has been considered to have multiple roles including plant defense against pests and diseases (Amtmann et al., 2008; He et al., 2020). K-starvation may have interfered with mechanisms related to plant disease resistance to F. oxysporum V5w2 resulting in poor growth.

Soil sterilization exerted various effects on nematode-fungus-banana root interactions that are not only related to the removal of beneficial and pathogenic microbes (Li et al., 2019), but also to other physico-chemical changes that may have been instigated by the high sterilization temperatures (De Deyn et al., 2004; Lees et al., 2018). In the presence of N, banana plants grown in non-sterile soil had low densities of R. similis that caused less death of roots, resulting in better growth in terms of biomass and size. Poor plant growth in sterilized soil may indicate the elimination of beneficial microbes in the endospheres and rhizospheres (Ochieno, 2010; Kaushal et al., 2020), including nematode-suppressive microbes, arbuscular mycorrhizal fungi (AMF), phosphate solubilizing microbes among other beneficial soil biota (Paliwoda and Mikiciuk, 2020; Van der Veken et al., 2021). However, the poor growth of banana plants in the steam-sterilized soil may also have alternative explanations. For instance, it is possible that soil sterilization led to decreased bioavailability of P through heat-driven sorption (Shaw et al., 1999; Sinegani and Sedri, 2011; Bai et al., 2017), or even made the fixed P more water-soluble and hence prone to loss through leaching (Shaw et al., 1999; Sinegani and Sedri, 2011; Bai et al., 2017), with the condition being made more extreme through supply of the P-deficient solution. Despite the explained presence of beneficial microbes, root necrosis was higher in P-starved banana plants grown in non-sterile soil. This raises questions as to why root necrosis did not relate directly with R. similis densities and root death that instead appeared to have been suppressed. Evidence from the data presented in the present study shows that R. similis and soil sterility exerted independent activities toward root necrosis under the P-deficient conditions. It is likely that other root parasitic nematodes and root-rot microbes in non-sterile soil contributed to the development of the necrotic lesions under the P-limited conditions (Ochieno, 2010; Finkel et al., 2019). This may have involved the secretion of microbial phosphatases for the acquisition of P from disintegrating root tissues (Tarafdar et al., 2001; Cabugao et al., 2017, 2021).

Despite all these speculations having considerably valid bases, consensus toward the outcomes of interactions between F. oxysporum V5w2 and other fungal phytopathogens with plant-parasitic nematodes in the banana rhizosphere context (Ochieno, 2020), are complex matters that still exist within a conundrum of diverse views in plant sciences (Schulz and Boyle, 2006; Ochieno, 2010, 2020; Avedi et al., 2014; Taylor, 2014; Schouten, 2016; Brader et al., 2017; Maryani et al., 2019; Geiser et al., 2020; Ochieno et al., 2021). This requires more investment toward research on the diversity and function of root and rhizosphere microbiomes under different ecological conditions (Li et al., 2019; Berg et al., 2020; Kaushal et al., 2020). Proper identification of F. oxysporum V5w2 among other strains that have been proposed for biological control requires whole genome sequence (WGS) analysis to differentiate them from pathogenic ones within the Fusarium of Banana Complex (FOBC; Kurtz, 2010; Schouten, 2016; Maryani et al., 2019; Bedoya et al., 2021). Characterization of interacting endosphere and rhizosphere microbes and invertebrates could be strengthened with better methods such as those based on metagenomic DNA sequence analyses (Sharpton, 2014; Nesme et al., 2016; Zakeel and Safeena, 2020). Metabolomic analyses of root exudates based on gas chromatography-mass spectrometry (GC-MS), liquid chromatography (LC) platforms or nuclear magnetic resonance (H-NMR) would have provided further details on changes instigated due to banana root interactions with rhizosphere biota and nutrients among other factors (van Dam and Bouwmeester, 2016; Vives-Peris et al., 2020). Changes in rhizosphere microbe populations in the form of colony forming units (CFU) could be monitored through plate dilution method of plant and soil samples (Vieira and Nahas, 2005), or by other multiple culture-independent measurements including adenosine tri-phosphate (ATP), flow cytometry (FCM), quantitative real-time PCR (qPCR), phospholipid fatty acids (PLFA), and microbial biomass carbon (MBC) (Li et al., 2008; Zhang et al., 2017). Follow-up studies may as well explore the possible mechanisms of R. similis suppression in the non-sterile rhizosphere, which may include direct parasitism or predation on the nematode or its eggs (Sikora et al., 2008; Li et al., 2015), antibiosis through production of toxic metabolites (Mwaura et al., 2010; Keswani et al., 2020), competition for nutrients and other resources (Kerry, 2000), as well as induced systemic resistance (Sikora et al., 2008; Walters, 2009; Paparu et al., 2013; Poveda et al., 2020). Such studies may be complemented with those assessing microorganisms that assist the survival and parasitism of plant-parasitic nematodes by protecting them against antagonistic microbes (Topalović and Vestergård, 2021). It should also be considered that microbes such as arbuscular mycorrhizal (AM) fungi known to be beneficial to host plants may also suppress plant growth while increasing infections by plant-parasitic nematodes (Frew et al., 2018). In all these, it remains an open question on whether development and evaluation of resistant banana cultivars should focus on independent pest species (Tripathi et al., 2015; Seenivasan, 2017; Rebouças et al., 2018), or whether a broader approach that tries to simultaneously tackle disease complexes and abiotic stresses would be achievable in providing better banana cultivars (Lorenzen et al., 2010; Almeida et al., 2018; Batte et al., 2019; Tripathi et al., 2019, 2020; Rocha et al., 2020).

In summary, results from the present three experiments have shown that soil microbes provide ecosystem services through pest and disease suppression while promoting banana plant growth. Soil sterilization and tissue culture eliminates such beneficial microbes to enhance root infection and damage by R. similis and F. oxysporum V5w2. Plant growth promoting benefits of the soil microbes are negated by nutrient stresses, especially N deficiency that shifts their activities toward being competitors that suppress banana plant growth. However, under such extreme N-deficient conditions, pathogenic F. oxysporum V5w2 and R. similis may create some yet to be clearly understood impression of banana plant growth promoting services. Competition for N under extreme starvation of this very important nutrient explains the suppression of R. similis by F. oxysporum V5w2, but not the widely claimed induced systemic resistance mechanisms in banana plants. On the other hand, P deficiency appears to provide physiological conditions that favor synergistic interactions between F. oxysporum V5w2 and R. similis, whereby the densities of both pathogens in banana root tissues are increased. Potassium deficiency appears to provide physiological conditions that favor pathogenicity of F. oxysporum V5w2 by constraining plant growth, especially in terms of leaf development. These are part of issues that could inform the application of microbial services in Sustainable Food Systems (Timmis et al., 2017), particularly in the pending decision on registration of F. oxysporum V5w2 as a commercial biopesticide (Dubois et al., 2006, 2011; zum Felde, 2011; Ochieno, 2020, 2021; Kisaakye et al., 2022), in line with International Standards for Phytosanitary Measures (ISPM) among other related policies (Wabule et al., 2003; Avedi et al., 2014; KEPHIS, 2014; FAO-ISPM, 2021).

In conclusion, pathogenic interactions between R. similis and F. oxysporum V5w2 are predominantly suppressed by a complex of soil microbes that exert plant growth promoting effects in tissue culture banana plants through N, P, and K dependent processes. Nitrogen is the most important limiting factor in rhizosphere interactions between banana roots, beneficial microbes and the pathogens. Soil sterilization and the stringent aseptic tissue culture techniques still require the development of alternative innovative ways of conserving microbial services for sustainable agriculture.

The datasets presented in this article are not readily available because the conflict of interest as declared only makes it possible to refer to data that was jointly published in Ochieno (2010). Requests to access the datasets should be directed to dwochieno@gmail.com.

The author confirms being the sole contributor of this work and has approved it for publication.

This work was part of my PhD studies that were funded by Wageningen University and Research Center (WUR) (Project number PE&RC 06034) and the International Institute of Tropical Agriculture (IITA-Uganda) through finances from the German Federal Ministry for Economic Cooperation and Development (BMZ) (GTZ Project number 03.7860.4-001.00). Masinde Muliro University of Science and Technology (MMUST) under the leadership of the Vice Chancellor among other staff provided funds for presentation of this research at the X International Symposium on Banana ISHS-ProMusa Symposium, 10–14 October 2016, Montpellier, France.

This work has been disassociated from studies that propose Fusarium oxysporum V5w2 as an Endophytic Biological Control Agent and the matter has already been openly declared (Ochieno, 2020). Prof. Marcel Dicke, Prof. Arnold van Huis, Dr. Thomas Dubois, Dr. Danny Coyne and Dr. Piet J.A. van Asten have renounced their co-authorship due to difference of opinion about the interpretation of the data and the conclusions of this study. My current employer who is Masinde Muliro University of Science and Technology (MMUST) with its Centre for African Medicinal Flora and Fauna (CAMNFF) was not involved in the research described in this article.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.