Growth And Nutrition Of Cowpea (Vigna Unguiculata) Under .

Rhizophagus irregularis PH5 (RIcoat); Pseudomonas putida (PPcoat)and a mix of R. irregularis P. putida (MIXcoat)] and three waterregimes [no water deficit, 80–75% of water holding capacity2.6 Biomass production, seed yield and nutrientsacquisition(D0); moderate water deficit, 60–55% of water holding capacityAt harvest, pods were separated and weighted to determine fresh(D1); and severe water deficit, 30–25% of water holding capacityweights. After recording the weight of pods, seeds were collected(D2)]. Each treatment had six replicates. During the first 3 weeksand weighted. Shoots and roots were dried for 2 days at 75 C toof plant growth, water was supplied daily to reach 80% of waterobtain dry weights. Seed yield was calculated by multiplying theholding capacity in all treatments. Volumetric soil moisture wasnumber of pod per plant by the number of seeds per pod and themeasured with a ML2x ThetaProbe (AT Delta- T Devices Ltd,seed weight mean (Sinha, 1977). After drying, shoots were grindedCambridge, UK), where changes in the apparent dielectric con-and digested according to the European Standard EN 13805stant of moist soil allowed measuring the volumetric soil moisture(2014). A segmented flow analyser was used for total N evaluationcontent (Roth, Malicki, & Plagge, 1992; White, Knight, Zegelin, &(Skalar Inc. SanPlus, The Netherlands) and inductively coupledTopp, 1994). Before starting the experiment, measures were per-plasma optical emission spectrometry (ICP- O ES; GBC Quantima,formed to match the water holding capacity of the soil with theAustralia) for total P and K. The ICP- O ES operating conditionsvolumetric soil moisture. The 100%, 85–80%, 60–55% and 30–were as follows: 1,000 W RF power—1,000 W, 15.0 L/min plasma25% of soil water holding capacity corresponded to 22, 16, 10–9gas flow rate, 1.2 L/min auxiliary gas flow rate, 1.0 L/min carrierand 6–5% volumetric soil moisture, respectively. In order to con-gas flow rate, 50 scan/reading, 3 measurement replicates and dualtrol water deficit and maintain it at the desire level, the soil waterdetector.content was measured daily with the ThetaProbe ML2x at the endof the afternoon (5:00–6:00 p.m.) and the amount of water lostwas added to each pot. For fertilisation, each plant received 20 ml2.7 Mycorrhizal developmentof modified white mineral solution P2N3 (Gryndler, Vejsadová, &Mycorrhizal colonisation in the roots of cowpea was assessed by mi-Vančura, 1992) twice a week.croscopic methods. The roots were carefully washed and stained asdescribed in a modified Phillips and Hayman (1970) protocol (Oliveira,2.4 Gas exchange parametersThe steady- s tate net photosynthesis A (Pn), stomatal conductanceVosátka, Dodd, & Castro, 2005). The percentage of root length colonised (RLC) was evaluated by the grid- line intersect method (Giovannetti& Mosse, 1980) under a stereomicroscope (Leica EZ4 HD, Germany).(gs), intercellular CO2 concentration (Ci) and transpiration rate (Tr)were determined using a Li- 6 400 IRGA (LI- COR, Lincoln, NE, USA).A 300 μmol/s flow of non- contaminated air was provided to the2.8 Statistical analysisleaves using a leaf chamber and mass flow controllers. The ana-Normality and homogeneity of variances were confirmed and datalysed leaves were exposed to a saturating photosynthetic photonanalysed by one- way and two- way analysis of variance (ANOVA) forflux density of 1000 μmol m 2 s 1, block leaf temperature of 25 Ceach dependent variable versus the independent variables (inocula-and with the relative humidity of the air within the apparatus rang-tion and water regime). In some cases, transformation was performeding between 45 and 55%. In all cases, only mature, fully expandedbefore analysis, to normalise skewed distributions before ANOVA.leaves were selected for measurements from four different plantsThis was the case of data of mycorrhizal colonisation (x2), N shootof each experimental condition. The measurements for gas ex-concentration (1/x), stomatal conductance (x1/3), transpiration ratechange were recorded between the late morning (9:00–11:00 a.m.)( x), water use efficiency (1/x) and carotenoids leaf content ( x). Theand early afternoon (1:00–3:00 p.m.). The instantaneous water usemain effects of the factors inoculation (Control, PPcoat, RIcoat andefficiency (WUE) (μmol CO2 per mmol H2O) was calculated by divid-MIXcoat), water regime (D0, D1 and D2) and their interaction wereing the values of steady- s tate net photosynthesis by the transpira-analysed. When a significant F- value was obtained (p 0.05), treat-tion rate (Pn/Tr).ment means were compared using Duncan's multiple range test. Allstatistical analyses were performed with the SPSS 25.0.0 software2.5 Chlorophylls and carotenoids contentFresh cowpea leaves (about 0.2 g) were homogenised in chilledN, N- dimethylformamide and stored overnight in the dark at4 C (Moran & Porath, 1980). The absorptions were measuredat 664, 647 and 461 nm using a HACH DR/4000U spectropho-package (IBM SPSS Statistics, USA).3 R E S U LT S3.1 Plant growth, yield and nutrients concentrationtometer (HACH Company, Loveland, CO, USA). Chlorophyll a andSeeds coated with R. irregularis inoculum (singly or mix) took ap-chlorophyll b were estimated using the equations of Inskeep andproximately 7 days to final emergence from the soil, while thoseBloom (1985) and carotenoids using the equation of Chamovitz,inoculated with bacteria and control took 4 days. Shoots, rootsSandmann, and Hirschberg (1993).and total dry weights of cowpea were negatively affected by

TA B L E 1 Biomass production and seed yield of Vigna unguiculata (L.) Walp. under different inoculation treatments [non‐inoculated(Control) Rhizophagus irregularis (RIcoat), Pseudomonas putida (PPcoat) and the mix R. irregularis P. putida (MIXcoat)] and no water deficit(D0), moderate water deficit (D1) and severe water deficit (D2)InoculationWater regimeShoot dryweight (g)Root dryWeight (g)Total plant dryweight (g)Root/Shoot ratioSeed weight (g)Seed yield (g)ControlD01.2 0.1 cd0.9 0.1 e2.0 0.2 g0.8 0.0 abc0.07 0.0 ab0.4 0.1 aD10.7 0.0 b0.5 0.1 cd1.2 0.1 def0.7 0.1 ab0.10 0.0 ab0.5 0.1 aD20.4 0.0 a0.3 0.1 abc0.8 0.1 bc0.8 0.1 bc0.08 0.0 ab0.2 0.1 aPPcoatRIcoatMIXcoatD01.3 0.1 d0.9 0.1 e2.1 0.1 g0.7 0.0 ab0.22 0.0 c1.0 0.2 cD10.8 0.0 b0.6 0.1 d1.4 0.1 ef0.8 0.1 bc0.15 0.0 bc0.9 0.0 bcD20.4 0.0 a0.5 0.1 cd0.9 0.1 bcd1.1 0.2 c0.12 0.0 abc0.4 0.1 aD01.1 0.1 c0.4 0.0 bcd1.5 0.1 f0.4 0.0 a0.06 0.0 a0.3 0.2 aD10.6 0.1 b0.2 0.0 ab0.8 0.1 bc0.4 0.1 ab0.08 0.0 ab0.4 0.1 aD20.3 0.1 a0.1 0.0 a0.4 0.1 a0.6 0.2 ab0.07 0.0 ab0.2 0.1 aD01.0 0.0 c0.4 0.1 bcd1.4 0.1 ef0.4 0.1 a0.05 0.0 a0.4 0.1 aD10.8 0.1 b0.3 0.1 abc1.1 0.1 cde0.5 0.1 ab0.11 0.0 ab0.5 0.1 abD20.4 0.1 a0.3 0.1 abc0.7 0.2 ab0.6 0.2 ab0.05 0.0 a0.2 0.1 aNote. Means ( 1 SE) followed by letters that indicate significant differences between treatments according to Duncan's multiple range test at p 0.05.water regime, especially by severe water deficit (Tables 1 and 2).treatments and consequently inferior values of root biomass overIn general, the roots and total biomass were significantly affectedshoot (Table 2). The seed yield was significantly impaired by theby the inoculation treatments, positively by P. putida and nega-severe water deficit (Table 2). Inoculation and water regime hadtively by R. irregularis (Table 2). There was no significant effect ofsignificant main effects on cowpea shoot nutrients concentra-inoculation on shoot dry weight under the different water regimestion (Table 2). In general, the presence of R. irregularis increasedwhen compared with control (Table 1). Overall, PPcoat treatmentN and P shoot concentrations when compared with controlhad a significant enhancement effect in total plant dry weight,(Table 2). Yet, the interaction between water regime and inocula-seed weight and seed yield of cowpea (Table 2). Under moderatetion showed only significant increase of N in plants under no waterwater deficit, plants inoculated with P. putida presented a signifi-deficit (Figure 1), with an increase of 38% in shoot concentration.cant increase in seed yield (Table 1). RIcoat treatments presentedComparing with the corresponding control, P shoot concentrationlower root biomass when compared with the PPcoat and controlwas significantly increased in the treatments of RIcoat D0, Mix D1TA B L E 2 Main effects of the factors inoculation and water regime and two‐way ANOVA F‐values and significances for biomassproduction, seed yield and nutrient shoot concentration of Vigna unguiculata (L.) WalpShoot dryweight (g)Main EffectsInoculation (I)Water regime(WR)Root dryWeight (g)Total plantdry weight(g)Root/ShootratioSeedweight (g)Seedyield (g)N (g kg 1)P (g kg 1)K (g kg 1)Control0.7 ab0.6 b1.3 b0.8 b0.1 a0.4 a12.3 b1.5 a25.3 aPPcoat0.8 b0.6 b1.5 c0.9 b0.2 b0.7 b9.3 a1.4 a27.3 aRIcoat0.7 a0.3 a0.9 a0.5 a0.1 a0.3 a14.7 c2.0 b26.1 aMIXcoat0.7 a0.3 a1.0 a0.5 a0.1 a0.4 a15.4 c2.3 c26.4 aD01.1 z0.6 z1.7 z0.6 x0.1 x0.5 y12.2 x1.8 x22.6 xD10.7 y0.4 y1.2 y0.6 x0.1 x0.6 y12.5 x1.6 x27.0 yD20.4 x0.3 x0.7 x0.8 y0.1 x0.2 x13.1 x1.8 x30.6 *Two‐way ANOVA F‐values and significancesInoculation (I)Water regime (WR)I WR3.8*140.5***1.2 ns24.0***1.5 ns85.5***3.8*1.4 ns8.9***0.9 ns2.9 ns41.0***1.5 ns0.5 ns1.1 ns1.1 ns0.9 ns3.8**1.0 ns,,Notes. Letters indicate significant differences according to Duncan's Multiple Range test. * ** ***significant effect at the level of p 0.05, p 0.01 andp 0.001, respectively; ns, non‐significant effect. Control, non‐inoculated; PPcoat, Pseudomonas putida; RIcoat, Rhizophagus irregularis; MIXcoat, mixof R. irregularis and P. putida; D0, no water deficit; D1, moderate water deficit; D2, severe water deficit.

N (g/kg)(a)25de2015edebcdcdedecdecdebcababa10No water deficit e water deficit (D1)Severe water deficit (D2)P at5bcbcdbcabaPPcoatabControl22cdedecdeabab1No water deficit tabcdRIcoataMIXcoataRIcoatababcRIcoatcd35201510No water deficit (D0)and D2 by 39%, 85% and 57%, respectively. The accumulation of Kin cowpea shoots was mainly affected by the water regime, beingModerate water deficit (D1)PPcoatControlPPcoatControl0PPcoat5ControlF I G U R E 1 Effects of differentinoculation treatments [non- inoculated(Control), with Rhizophagus irregularis(RIcoat), Pseudomonas putida (PPcoat) andthe mix R. irregularis P. putida (MIXcoat)]and water regimes on N (a), P (b) and K (c)shoot concentration in Vigna unguiculata(L.) Walp. Values are means 1 SE andletters indicate significant differences(p 0.05) according to Duncan's multiplerange testSevere water deficit (D2)K (g/kg)4025MIXcoatModerate water deficit (D1)(c)30RIcoatMIXcoatRIcoatControl0PPcoat1Severe water deficit (D2)3.2 Mycorrhizal root colonisationincreased by moderate and severe water deficits (Table 2). SinglyPlants without R. irregularis inoculation (control and P. putida inocula-inoculated P. putida improved K shoot concentration by 25% undertion) had no root mycorrhizal colonisation. Treatments where R. ir-moderate water deficit (Figure 1).regularis was inoculated had root colonisation that varied with water

RLC (%)70cbc6050ab40abab30a20100RIcoatMIXcoatNo water deficit (D0)RIcoatMIXcoatModerate water deficit (D1)RIcoatMIXcoatSevere water deficit (D2)F I G U R E 2 Percentage of root lengthcolonisation (% RLC) in the roots of Vignaunguiculata (L.) Walp. inoculated withRhizophagus irregularis (RIcoat) or themix R. irregularis Pseudomonas Putida(MIXcoat) via seed coating under differentwater regimes. Values are means 1 SEand letters indicate significant differences(p 0.05) according to Duncan's multiplerange testregime (Figure 2). Both moderated and severe water restrictionsvariation on gas exchange parameters such as photosynthesis, sto-negatively affected the presence of R. irregularis in the roots. Whenmatal conductance or transpiration imposed by water stress canno water deficit was imposed, the percentage of RLC was higherhamper plant growth (Farooq et al., 2008; Li et al., 2014), which wasthan 50%. Inoculation with P. putida did not have a significant impactshown in our results (Table 1 and Figure 3). Equally, water deficit sig-on root colonisation by R. irregularis.nificantly decreased the content of chlorophyll a, chlorophyll b andcarotenoids in cowpea leaves (Table 3). Photosynthetic pigments are3.3 Leaf parametersimportant for plants to harvest light and produce reducing powers.Carotenoids play a key role in plant antioxidant defence system byBoth water regime and microbial inoculation influenced cowpea leafquenching singlet oxygen and peroxyl radicals, protecting the pho-gas exchange parameters (Figure 3a–e and Table 3). Severe watertosynthetic tissue from oxidative damage (Jaleel et al., 2009).deficit negatively affected the gas exchange parameters in bothLegume crops are able to establish symbiotic interactions withnon- inoculated and inoculated treatments (Figure 3 and Table 3).microbes (e.g. PGPR and AM fungi), which help them cope with un-The presence of mycorrhiza singly and in combination with P. putidafavourable environmental conditions such as drought (Oliveira et al.,significantly enhanced Pn when no water deficit was imposed2017a,b; Zahran, 2010).(Figure 3a). Also, under no water deficit, the treatment MIXcoat pre-Cowpea is considered to be highly mycotrophic (Molla & Solaiman,sented higher values of gs and Tr (Figure 3b,d). Intercellular CO2 con-2009) which leads to enhancement of below and above ground bio-centration was adversely impacted by severe water deficit (Table 3).mass, nutrients accumulation, protein content and grain yield underPlants singly inoculated with P. putida showed the lower values ofdifferent water regimes (Kwapata & Hall, 1985; Oliveira et al., 2017a;Pn, gs and Tr in all water regimes. WUE (Figure 3e) was significantlyOruru, Njeru, Pasquet, & Runo, 2018; Rabie, Aboul- Nasr, & Al- higher in plants under severe water deficit and in the presence ofHumiany, 2005). However, our results showed that association be-microbial inoculants.tween AM fungi and cowpea did not result in increased plant growthChlorophyll and carotenoids varied according to microbial inocu-or seed yield (Tables 1 and 2). Moreover, for root weight and root/lation and water regime (Figure 4 and Table 3). Plants under moder-shoot ratio the values of plants inoculated with R. irregularis wereate and severe water deficit had significantly lower concentrationslower than control. This can be related to the fact that the productionthe leaf pigments, irrespective of microbial inoculation (Table 3). Inof fungal mycelium is much more cost- effective in terms of organicgeneral, plants inoculated with R. irregularis enhanced both chloro-carbon (C) than the production of equivalent root length (Table 2).phylls and carotenoids contents, even under severe water deficit,Consequently, plants adjust belowground C allocation contributingwhen compared with PPcoat and control treatments (Table 3).to the formation of a shorter mycorrhizal root system (Jacobsen,Smith, & Smith, 2002), relying on the fungal mycelium for nutrient4 D I S CU S S I O Nuptake (Smith, 2000). In fact, there was a significant enhancementin shoot nutrient content (Table 2), particularly N and P, which hasalso been described in other studies with inoculated cowpea (Boby,The frequency and intensity of drought can dramatically decreaseBalakrishna, & Bagyaraj, 2008; Oruru et al., 2018; Sanginga, Lyasse,plant biomass and grain yield (Farooq et al., 2017). Ahmed and& Singh, 2000; Yaseen, Burni, & Hussain, 2011). Still, this enhance-Suliman (2010) showed cowpea yield reductions of 34–66% underment in nutrient content was not enough to result in greater yields,water stress during the reproductive stage of crop development,fact perhaps associated with the sink of carbohydrates of the fun-and Akyeampong (1985) revealed 29% of declination during podgal mycelium that the plant could not allocate to seed developmentfilling. Our results showed that both moderate and severe waterand filling. Also, the observed delay on seedling emergence of plantsdeficit decreased shoots, roots and total biomass and that severeinoculated with AM fungi might have a negative influence on cow-water deficit significantly reduced seed yield (Table 2). The negativepea yield or even adaptation to the water deficit. Faster germination

Photosynthetic rate A (P n )µmol CO2 m–2s–1(a)8060fefdded50c40decbcb30a20No water deficit IXcoatPPcoatMIXcoatRIcoatPPcoatControlSevere water deficit rolddcd150100No water deficit 50ControlF I G U R E 3 Effects of microbialinoculation [non- inoculated (Control),Rhizophagus irregularis (RIcoat),Pseudomonas putida (PPcoat) and themix of R. irregularis P. putida (MIXcoat)]and water regime on Pn (a), gs (b), Ci (c),Tr, (d) and WUE (e) of Vigna unguiculata(L.) Walp. Letters indicate significantdifferences (p 0.05) according toDuncan's multiple range testModerate water deficit (D1)Intercellular CO2 concentration (Ci)µmol CO2 /mol(c)250ControlMIXcoatRIcoatPPcoatNo water deficit olSevere water deficit (D2)RIcoat1.00Moderate water deficit (D1)Stomatal conductance (gs)mol H2 O m–2 coataMIXcoat70Moderate water deficit (D1)Severe water deficit (D2)and establishment increases the opportunity of seedlings to achievein R. irregularis- inoculated plants (Table 3), particularly under se-a positive C and nutrient balance, which is crucial, especially undervere water deficit for chlorophyll a and b (Figure 4). WUE, one ofstress conditions (de Albuquerque & de Carvalho, 2003). Furtherthe mechanisms of plants to increase drought resistance (Vivas,studies are, therefore, needed to improve this limitation on the ger-Marulanda, Ruiz- Lozano, Barea, & Azcón, 2003), was increased inmination of cowpea seeds coated with AM fungi.plants inoculated with R. intraradices and P. putida under severeOn the other hand, when compared with control, there waswater deficit (Figure 3). The presence of mycorrhiza significantly en-an overall enhancement on chlorophyll and carotenoids contentshanced photosynthetic rate, stomatal conductance and transpiration

Transpiration rate E (Tr)mmol H2O m–2 s–1(d)f25ee15e10cdeedcd5bcbaNo water deficit (D0)Moderate water deficit (D1)Severe water deficit (D2)Water use efficiency (WUE)µmol CO2 per mmol PPcoatcdefControl5abRIcoat10PPcoat15No water deficit (D0)Moderate water deficit (D1)PPcoatMIXcoatControl0Severe water deficit (D2)FIGURE 3 Continuedrate (Figure 3) under no water deficit, corresponding to the wateret al., 2016). In our results, the co- i noculation (PGPR AM fungi)regime where the colonisation was higher (Abdel- Salam, Alatar, &apparently did not present any extra benefit to the plants. OnEl- Sheikh, 2017). The increased rate of photosynthesis was probablythe other hand, plants singly inoculated with P. putida showeda result of the increased use of fixed C (Fitter, 1991) and/or highera significant increase in seed yield (Table 2), including underchlorophyll content (Gusain, Singh, & Sharma, 2015), under no watermoderate water deficit (Table 1). Overall, P. putida significantlydeficit (Figures 3 and 4). Under severe water deficit, this relation-enhanced total plant biomass (Table 2). The accumulation of K inship between photosynthesis and chlorophyll content was not socowpea shoots was enhanced by 25% in plants singly inoculatedobvious. Water deficit affects various physiological and biochemicalwith P. putida under moderate water deficit (Figure 1). K is an es-processes of plants, limiting stomata and transpiration and result-sential nutrient for plants and plays an important role in droughting in reduced photosynthesis (Farooq, Wahid, Kobayashi, Fujita, &conditions, cell membrane stability, roo

Growth and nutrition of cowpea (Vigna unguiculata) under water deficit as influenced by microbial inoculation via seed coating Inês Rocha1 Ying Ma1 Miroslav Vosátka2 Helena Freitas1 Rui S. Oliveira1,3 1Centre for Functional Ecology – Science for People & the Planet, Department of Life Sciences, University of Coimbra, Coimbra, Portugal