Effects of vemicompost leachate (VCL), Kelpak® (KEL) and smoke-water (SW) on V. unguiculata morphological growth under different watering regimes after 13 weeks
Water limitations reduced 90% of the measured morphological parameters with reductions being more pronounced in control plants than biostimulant-treated plants. Reductions in the plant’s biometric characters occur under water deficits due to water/nutrient deprivation or high transpiration rate (Anjum et al. 2011). Thus, a widespread root system is a requisite to improve water extraction from shallow soil horizons before being lost to evaporation (Ashraf and Foolad 2005; Chinsamy et al. 2013). The significantly longer root length and widespread adventitious roots elicited by KEL and SW (Figs. 1B and 2) resulted in improved nutrient uptake and water availability under drought (Georgiev 2004; IIyas et al. 2021). This may have contributed to their augmented leaf growth and 3-fold increase in shoot length. Water and nutrient availability regulate shoot length increase. Among the required nutrients is N2 that increases nodes and water increases the internodal distances (Maleki et al. 2013), nutrient hydrolysis and averts dehydration. Furthermore, an enzyme known as acid invertase is linked with plant growth (Sturm 1999). The increased invertase activities in biometric characters of primed chickpea (Cicer arietinum cv. GPF-2) elevated hexose supply which may have increased energy and growth of the above-ground biomass (Kaur et al. 2005).
Root-to-shoot signaling is a prominent event that causes stomatal closure under drought stress via ABA increase which prevents further water loss to the transpiration stream (Barnabás et al. 2008). However, stomatal closure prevents CO2 fixation, decreasing leaf internal CO2 and promotes pentadiulose-1,5-bisphosphate (Rubisco inhibitor) and photorespiration, eventually culminating to a reduction in photosynthesis rate (Parry et al. 1993; Schafleitner et al. 2013; Daryanto et al. 2015). Hence, the increased root system caused by biostimulants in Fig. 2 could mean that biostimulants capacitated the less watered plants with growth promotors to curtail root-to-shoot stress signal. This may have played a major role in the increasing of shoot length, number of leaves and leaf area (Fig. 1A and C; Table 1) to prioritize vegetative growth, light interception and CO2 absorbability. KEL and VCL down-regulated the production of ABA and increased photosynthesis activity, growth and survival rate of Ceratotheca triloba (Masondo 2017). García et al. (2014) showed that the capacity of biostimulants to alleviate stress during ABA biosynthesis is via ABA independent metabolic pathways. Therefore, the capability of VCL and KEL to curb ABA biosynthesis could be similar to exogenous applications which induces active plant growth (Stirk et al. 2014; Aremu et al. 2015a, b; Kocira et al. 2020). Moreover, the present study indicates that limiting watering frequency to once-a-week improved the number of nodules in SW- and VCL-treated plants by almost 2- and 4-fold, respectively (Fig. 1D), translating into an enhanced N modulation.
Effects of vemicompost leachate (VCL), Kelpak® (KEL) and smoke-water (SW) on V. unguiculata flowering under different watering regimes after 3 weeks.
Drought impairs yield before and post-anthesis (Nadeem et al. 2019). At flowering and anthesis, drought reduces yield by 27-57% in chickpea (C. arietinum), common bean (Phaseolus vulgaris) and mungbean (Vigna radiata) (Rosales-Serna et al. 2004; Mafakheri et al. 2010; Ahmad et al. 2015). The present study showed that water deprivation can reduce flowering in cowpea by 69%, depending on biostimulant and irrigation regime. For instance, only flowers of SW-plants were improved considerably under once-a-week regime. Conversely, raising watering frequency to twice- and thrice-a-week increased flowers in all biostimulant plants by more than 2-fold. Biostimulants also induced more immediate anthesis in all watering regimes, demonstrating efficacy against inhibitory effects of water stress reported by Barnabás et al. (2008).
Physiological effects of biostimulants on V. unguiculata biochemicals under different watering regimes.
Disruptive influences of drought alter sugar metabolism, phloem loading mechanisms and reduces leaf growth while roots continue to grow due to less water sensitivity and high sink demand (Lemoine et al. 2013). Sucrose and hexose levels shift by increasing, whereas starch contents decline (Pelleschi et al. 1997). Such biochemical changes are used as an indicator of induced sucrose synthesis and starch hydrolysis (Lemoine et al. 2013). Restricting irrigation to once a week slightly improved leaf carbohydrates of biostimulant-treated plants and significantly raised carbohydrates of roots of SW and control plants (Fig. 3A). In cotton (Gossypium hirsutum), accrued sucrose and hexose act as an energy supply to safeguard cells or for adjustment for osmosis under adverse conditions (Burke 2007). Tripling water availability induced 2-fold increase in foliage carbohydrates of VCL plants, demonstrating its promotory effects under water stress and favourable conditions (Fig. 3C). These results disagree with the findings of Ngoroyemoto et al. (2019; 2020) and Bidabadi et al. (2016) where carbohydrates of Amaranthus hybridus and Stevia rebaudiana did not differ significantly following VCL application. This could be due to the efficacy of biostimulants depending on the type of biostimulant, plant species or cultivar (Du Jardin 2015). In seaweed extracts and VCL, this may further depend on the growth stage, application mode, extraction method and algae/earthworm species (Radovich and Norman-Aranco 2011; Battacharyya et al. 2015). Tomato (Solanum lycopersicon) exhibited susceptibility to drought via increased accumulation of proline, glucose, and sucrose (Nahar and Gretzmacher 2002). Similar findings have been reported by Chinsamy et al. (2013) in tomato seedlings under salt stress (which is common in drought-proned areas) but using VCL. This may be due to elevated root sink demand (Hummel et al. 2010), conversion of starch to sugars or reduced breakdown of stored starch by plant tissues (Chinsamy et al. 2013). More interestingly, root carbohydrates of the plants watered with VCL and KEL under the same water deficits were within the ranges of the plants watered twice and thrice per week, respectively (Fig. 3A, B and C). These results conform with the findings of Chinsamy et al. (2014) wherein carbohydrate content of VCL-treated tomato seedlings was increased with an increase in watering regimes but dropped significantly with water limitations.
As an addition to stomatal closure, increased cuticle thickening, plant cells accumulate soluble proteins, amino acids and alkaloids to establish osmotic adjustment (Reddy et al. 2004; Barnabás et al. 2008; Vurukonda et al. 2016; Ilyas et al. 2021). In the present study, restricting water application to once-a-week induced a significant increase of 4-fold in leaf proteins of control plants compared to when applied thrice-a-week (Fig. 3D and F). High concentrations of amino acids in water-stressed chickpea could have been due to protein hydrolysis (Ashraf and Iram 2005). According to Aranjuelo et al. (2011), water-stressed plants can partition significant amounts of C and N resources to promote leaf biosynthesis of osmoprotectants such as proline for osmotic adjustment and turgor maintenance. Ghanbari et al. (2013) reported high leaf N, proteins and proline content in drought-tolerant genotypes of Red beans and Chitti beans under water deficits. Interestingly, restricting watering frequency to once-a-week induced more than a 3-fold decline in leaf proteins in VCL- and KEL-treated plants (Fig. 3D). The decline was 5-fold in plants drenched with SW, indicating even higher leaf protein hydrolysis and /or translocation to sinks. The induced protein declines fall within the range of those found in plants watered thrice-a-week (Fig. 3F). A link between protein synthesis and C metabolism is integral to avert C starvation in developing tissues where protein synthesis contributes to the biosynthesis of new biomass (Smith and Stitt 2007; Piques et al. 2009). VCL promoted this relationship between the two processes in S. rebaudiana (Bidabadi et al. 2016). Therefore, the significantly reduced leaf proteins in the least and highly watered biostimulant-plants could mean that their protein synthesis may have been harnessed for growth and biomass accumulation rather than synthesis of protein-based compatible solutes.
Physiological effects of biostimulants on V. unguiculata photosynthetic pigments under different watering regimes in the greenhouse.
Drought induces low water potential, disrupting plant and nutrient uptake which results in osmotic stress and promotion of Na+ ions that perturbs the cell ionic equilibrium; enzymatic functions, mitosis and reduces the rate of photosynthesis (Mahajan and Tuteja 2005). It also modifies chlorophyll pigments, proteins or even oxidation of lipids in chloroplasts (Menconi et al. 1995). Chlorophyll a and b are highly susceptible to water deficits (Farooq et al. 2009; Ghanbari et al. 2013). Decreased chlorophyll biomolecules in water-stressed plants is an indicator of oxidative stress caused by chlorophyll degradation and pigment photooxidation (Anjum et al. 2011). Confronted with this evidence, Cramer et al. (2011) reported high chlorophyll pigments in drought-tolerant transgenic maize (Zea mays). In the present study control plants watered once-a-week had 2-fold greater chlorophyll a with chlorophyl a + b and carotenoids remaining similar to that of the control plants watered thrice-a-week. These findings suggest a remarkable dehydration-threshold in cowpea as reported by Clark (2007), Cruz et al. (2014) and Masenya (2016). Moreover, chlorophyll a, a + b and carotenoids increased immensely under once- and thrice-a-week owed to biostimulant applications which translates into greater improvement of photosynthesis (Table 2). KEL and VCL significantly enhanced chlorophyll a, b and a + b of A. hybridus (Ngoroyemoto et al. 2019). KEL replenished depleted photosynthetic pigments, proteins and carbohydrates in A. hybridus inoculated with Bacillus licheniformis and Pseudomonas fluorescens, thus improving photosynthetic activity, protein synthesis (Ngoroyemoto et al. 2020) and carboxylation. In response to abiotic stress, seaweed extracts promote chloroplast biogenesis and delay senescence (Battacharyya et al. 2015). Jannin et al. (2013) observed down-regulation of cysteine proteases linked with senescence and up-regulation of genes associated with stress response, increased photosynthesis, N and metabolism following Ascophyllum nodosum application. Cytokinins in KEL and VCL protect chloroplasts, membranes, increase chloroplast division (Arthur et al. 2001; Stirk and van Staden 2006; Battacharyya et al. 2015) and promote regulation of stress-related phytohormones and water equilibrium (Ilyas et al. 2021).
Physiological effects of biostimulants on V. unguiculata total phenolics and flavonoids under different watering regimes in the greenhouse.
Phenolics accrue in plant tissue as tannins, flavonoids and lignin precursors, to scavenge toxic ROS induced by drought stress (Rice-Evans et al. 1997; Battacharyya et al. 2015). In the present study, leaves of all plants accumulated greater phenolic concentrations relative to roots. Restricting watering regime to once-a-week significantly increased leaf phenolics in control plants compared to KEL-treated plants (Fig. 4A). The increased phenolic compounds are ascribed to stress-induced disruptions in metabolic processes of the cells (Keutgen and Pawelzik 2009) which may have been down-regulated in KEL-treated plants using an independent antioxidant pathways. Although ABA regulates various morpho-physiological processes to acclimatize plants to abiotic stresses (Ilyas et al. 2021), the capacity of biostimulants to alleviate stress during ABA biosynthesis is via ABA independent metabolic pathways (García et al. 2014). Therefore, the capability of KEL to curb ABA biosynthesis can be associated with its exogenous application which introduces active plant growth regulators to the plant (Stirk et al. 2014; Kocira et al. 2020). Additionally, SW induced marked declines in phenolics under similar water deficits as KEL (Fig. 4A). This may be linked to inhibitory properties of trimethylbutenolide (3,4,5-trimethylfuran-2(5H)-one) or stress-related growth regulators of SW reported by Kulkarni et al. (2008, 2011).
Flavonoids activate better plant response to abiotic stresses including drought (Battacharyya et al. 2015). In the present study, leaf flavonoids in water-stressed plants drenched with biostimulants decreased considerably, reaching significant concentrations in VCL-treated plants (Fig. 4D). There was also a decreasing ‘mirror-image’ trend in root flavonoids of biostimulant-treated plants as drenching frequency was reduced from thrice- to once-a-week. These findings suggested an inhibition in cowpea’s susceptibility to drought. Flavonoids alleviate stress by acting on enzymes, metabolic pathways (Araújo et al. 2008) and as cell protectants against apoptosis (Ndhlala et al. 2010). Enzymatic activity of chalcone isomerase increased following seaweed extract application (Battacharyya et al. 2015). Chalcone isomerase catalyses the biosynthesis of flavanone precursors and phenylpropanoid protective compounds (Sun et al. 2019).