Nutritional status of Zombi pea (Vigna vexillata) as influenced by plant density and deblossoming

Feeding billions, a healthy and nutritious diet in the era of climate change is a major challenge before plant breeders, geneticists and agronomist. In this context, the continuous search for adaptive and nutritious crops could be a better alternative to combat the problems of hunger and malnutrition. The zombi pea, a nutritious and underutilized leguminous vegetable, is one of such better alternatives to feed billions a nutritious food besides being a potential gene source for breeding abiotic stress resistant varieties. To evaluate its potential as a wonder crop in the tropical and subtropical regions of India, the nutritional status of tubers, pods and pericarp were investigated under different treatments of plant spacings and deblossoming. The experiment was conducted in split plot design with three replications and eight treatments during 2021–2022 in the coastal regions of India. The nutrient profiling in tubers and pericarp of pods in zombi pea revealed higher accumulation of nutrients viz. potassium (K), magnesium (Mg), iron (Fe), manganese (Mn) and zinc (Zn) with blossom retention. The zombi pea tubers reflected significantly high protein accumulation with the increase in plant spacing. The results pertaining to nutrient profiling in the pods of zombi pea indicated that the plant spacing has no significant effect on the accumulation of majority of nutrients under study. The above-mentioned findings are conspicuously novel and valuable. The present study would pave the way for understanding nutritional importance and breeding potential of this orphan crop. The blossom retention renders higher nutrient accumulation in tubers, pods and pericarp of zombi pea. Deblossoming has no significant influence on nutritional profile of this wonder crop but, wider spacing is effective in producing tubers with high protein content.


Experimental location and plant material
The field experiment was conducted during the 2021-2022 at the Regional Centre of ICAR-Central Tuber Crops Research Institute (CTCRI) (20.1450•N and 85.4706• E), Dumduma, Bhubaneswar, Odisha, India.The estimation of nutritional traits was carried out at the Charles Renard Analytical Laboratory (CRAL), ICRISAT.Growth parameter analysis was carried out at the Siksha 'O' Anusandhan (deemed to be university), Bhubaneswar, Odisha.The plant material comprised of Zombi pea line 'IC 259504' which is indeterminate in growth habit and was provided by ICAR-CTCRI, Regional Centre, Bhubaneswar and is registered at ICAR-National Bureau of Plant Genetic Resources (NBPGR), New Delhi.The material can be accessed from ICAR-NBPGR and is a cultivated form of Zombi pea.The field work was done at ICAR-CTCRI, Regional Centre only.

Experimental design and nutritional traits under study
The experiment was conducted in the Split plot design with 8 treatments and 3 replications.There were 4 levels of spacings (S 1 -45 cm × 15 cm, S 2 -45 cm × 30 cm, S 3 -60 cm × 15 cm and S 4 -60 cm × 30 cm) in the main plot and 2 levels of deblossoming (F 1 -flower removal and F 2 -flower retention) treatments in the sub plot (Table 1) The plan of layout is depicted in Table 2.All the flowers were removed during deblossoming (F 1 ) manually 60 days after sowing till peak flowering period.The sulfuric acid-selenium digestion approach as suggested by Sahrawat et al. 29 with minor modifications was employed for the analysis of protein, micro nutrients (Fe, Mn, Zn, Cu), macro (N, P, K) and secondary nutrients (Ca, Mg) in the tuber, pod and pericarp of zombi pea at ICRISAT Hyderabad, India.For the estimation of different nutrient levels in single digestion, sulfuric acid-selenium digestion mixture was utilized.Starch and sugar analyses were done as per the procedure presented by Stickland and Wilson 30 and Weng et al. 31 with minor modifications.

Sampling technique and nutrient analysis
Five plants from each plot were taken randomly excluding border plants and were tagged as sample plants for recording observations.About 0.5 g finely ground plant samples were taken in 250 ml digestion tube.Then, 14 ml of concentrated sulfuric acid comprising 0.5% selenium powder was immersed into each of the digestion tube to soak the grounded plant samples.For the preparation of Sulfuric acid-Se mixture, Se powder was dissolved in concentrated sulfuric acid by heating on a hot plate with intermittent stirring with a glass rod.The mixture was allowed to cool and final volume was made up to one litre.The digestion tubes were shifted to preheated block digester and the full digestion takes about 2.5 h which is indicated by colourless and clear plant digests after digestion.In case of Ca estimation, the dilution of plant digests with 30-40 ml of distilled water by keeping overnight is practiced.This is done to avoid the insoluble calcium sulfate.All the plant nutrient analysis were performed in triplicate as results reported as average values were analysed 29 The digests volume was made up to 250 ml with distilled water.Then aliquots of digests were used for respective estimation of nutrients like distillation with NaOH for N determination, phosphovanadomolybdate colorimetric method for the estimation of P and atomic absorption spectrophotometer for the determination of micronutrients like K, Ca, Mg etc 29 .
The starch and sugar content were determined through Spectrophotometer by phenol-sulphuric acid reagent method as per the protocol depicted by Stickland and Wilson 30 and Weng et al. 26 with minor alterations.

Analysis of quality parameters of tubers in tuber cowpea
The results pertaining to influence of treatments under study on quality parameters of tubers in tuber cowpea are presented in Table 4.The results indicated that the planting of tuber cowpea at wider spacing exhibited higher protein percentage in the tubers than closer spacing (Table 4).The deblossoming of tuber cowpea resulted in higher accumulation of proteins in the tubers (Table 4).The results indicated that starch and sugar content in tubers were not significantly influenced by variable spacings (Table 4).No significant variations were found with respect to interaction effects of treatments on different quality parameters of tubers in tuber cowpea (Table 4).
Table 3. Influence of spacing and deblossoming on nutritional value of tubers in tuber cowpea.www.nature.com/scientificreports/

Influence of spacing and deblossoming on nutritional value of green pods in tuber cowpea
The results presented in Table 5 indicated that the total N and P contents in pods were found to be independent of spacing treatments.The spacings S 1 (45 cm × 15 cm) and S 3 (60 cm × 15 cm) depicted significantly higher total K content (0.61, 0.61) than spacings S 2 (45 cmx 30 cm) and S 4 (60 cm × 30 cm).Ca, Mg, Fe, Mn and Zn contents also exhibited no significant variations at variable spacings (Table 5).At spacing of S 3 (60 cm × 15 cm), there was significantly higher Cu content (3.29) in the pods than at other spacings (Table 5).In general, the quality parameters were found to be independent of variable spacings in tuber cowpea.Similarly, the contents of majority of micronutrients under study were not affected by interactions of spacing and deblossoming treatments (Table 5).

Influence of spacing and deblossoming on protein content (%) of pods in tuber cowpea
The results pertaining to influence of treatments under study on protein content (%) of pod in tuber cowpea are presented in Table 6.The results indicated that protein contents in pods were not significantly influenced by variable spacings (Table 6).Greater accumulation of protein was found in F2 (blossom retention) (Table 6).Presence of blossoms may have been instrumental in greater accumulation of protein in the pods.The present findings are in agreement with those of Hussain and Basahy 33 , Gerrano et al. 34 , and Jayathilake et al. 35 .

Influence of spacing and deblossoming on nutrient content of pericarp in tuber cowpea
The results pertaining to influence of treatments under study on nutritional parameters of pericarp in tuber cowpea are presented in Table 7.The results revealed that total N, P, K, Ca, Fe, Mn, Zn, Cu content in pericarp was found independent of spacing treatments in the study (Table 7).Greater accumulation of N, P, K, Ca, Mg Fe, Mn, Zn, Cu was found under blossom retention conditions (F 2 ) (Table 7) The interaction effects of S 1 F 1 in the pericarp indicated higher accumulation of Mg (Table 7).

Influence of spacing and deblossoming on protein (%) of pericarp in tuber cowpea
The results pertaining to influence of treatments under study on the Protein % of pericarp in the pods of tuber cowpea are presented in Table 8.The results depicted no significant effect on the protein content of pericarp under variable spacing.However, the higher value of total protein accumulation in the pericarp was observed in F 2 treatment (6.63%) (Table 8).While interaction treatments have also exhibited no significant effect on pericarp protein accumulation (Table 8).

Soil parameters affected by tuber cowpea
Initial soil analysis showed pH-5.63,EC-0.

Correlation analysis
The results pertaining to correlation analysis among plant spacings and nutrient components have been presented in Tables 10, 11 and Fig. 2. The interaction analysis revealed the positive association between wide spacing and nitrogen (N), phosphorus (P), Manganese (Mn) and protein content of tubers under deblossoming condition.When flowers were not removed, the positive association was observed among area and N, P, Zn, Mn, Ca, Starch, Sugar and protein content.Under both conditions the plant spacing was negatively associated with potassium content.

Discussion
The flowers are the essential plant organs influencing nutritional composition, rate of sugar and dry matter accumulation, fruit quality traits, and physiological processes in the plants 25 .In the tuber cowpea, the simultaneous formation of tubers and flowers occurs, and the substances accumulated are transported to these organs, flowers, and tubers, respectively.Hence, the nutritional status of pods, pericarp and tubers was compared in the present investigation under different conditions.The total N and P content of tubers increased with the increase in plant spacing of tuber cowpea.Maximum nitrogen concentration was observed at the high spacing of tuberose 36 .In rice, higher P uptake was reported by Parvin et al. 37 at wider spacing 37 .Higher Zn and Mg contents of tubers were also observed at the widest spacing.At wider spacings, plants can absorb more water and nutrients, mainly because each plant produces a greater number of roots and has access to relatively greater proportions of soil and nutrients than a closely spaced plant.The highest zinc uptake in rice was also observed with the widest spacing, while the lowest uptake was recorded with the closest spacing 38 .Similar findings were reported by other researchers 39,40 .Magnesium is largely involved in chlorophyll synthesis, production, transportation, utilization of photo-assimilates, enzyme activation and protein synthesis 41 .Therefore, the higher protein content of tubers at wider spacing is also attributable to the higher content of Mg in tuber harvested from widely spaced plants.
In the present study, the wider row spacing of 60 cm was significantly better than 45 cm with respect to tuber protein content.The relatively higher levels of zinc observed in the tubers harvested from widely spaced plants, may also have been instrumental in increasing the protein content of such tubers, since zinc is known to be an  43,44 .The range of starch recorded in tuber cowpea is similar to that observed by other researchers [45][46][47] .The accumulation of more dry matter in the tuberous roots was probably accomplished by translocation of reserve carbohydrates from other plant parts.Under the deblossoming conditions, the inhibitory effects of flower and pod development on vegetative growth and tuber yield of zombi pea might be due to direct competition for photosynthates linked food reserves between different plant organs 25 .The results obtained in the present investigation substantiate this hypothesis.In general, the flower initiation process removes a huge quantity of nitrogen and carbohydrates.The consumed nitrogen is exploited in the development of pods and seeds 27,48 .The interaction analysis revealed that plant spacing was positively correlated with N, P and protein content in the tubers of zombi pea under deblossoming conditions.A large quantity of elaborated nitrogen and carbohydrates are required in tuber development.Thus, under wider spacing with deblossoming conditions, the more food material elaborated to tuber formation and enlargement in zombi pea, and resulting in well-developed tubers and a high quantitative accumulation of protein content in the tubers.Further, it might be supported by the results of Roberts and Struckmeyer 49 who recorded an increased phloem development in nonflowering plants as compared to flowering plants.In addition, the histological studies are required in zombi pea to analyze the alteration in blossoming and deblossoming conditions with respect to phloem development and flowering state.The mechanism involved when both tubers and flowers were produced in the same plant of Zombi pea can be explained by the fact that, there was unequal partitioning and translocation of assimilates involving those of carbon and of nitrogen.Larger proportions of carbon-based photo assimilate were translocated to the root tubers while greater amounts of nitrogen based assimilates were translocated to the fruits.It is possible that the inhibitory effects of floral and fruit development on vegetativeness and tuber development may be due to a simple direct competition for elaborated food materials between the vegetative organs, floral differentiation and fruit formation 24 .In legumes sucrose is the pre-dominant sugar and among nitrogenous solutes are the amino acids 50 .Sugars and other macromolecules arrive in heterotrophic plant tissues through the phloem 50 .Proteins are mobile in phloem and there may also be destination-selective translocation 31 .It was proposed that the difference in osmotic pressure between the phloem and the surrounding tissues could drive the unloading of solutes in sinks.
Forney and Breen 51 also reported that the starch content of roots of deblossomed strawberry plants was about 18 times higher than plants with fruit 51 .The contents of P, Ca, Zn, Mg, Fe, Cu in green pod were maximum in the treatment combination S3F2 (wider spacing with inflorescence retention).It is attributed to the fact that wider spacings allow each plant to produce more roots and get access to greater quantity of nutrients per unit area of soil.The relatively higher levels of copper observed in different plant parts like tuber, pod and pericarp at the closest spacing (S1) may be explained by the greater concentrations of copper recorded in the soil at the same spacing.Copper acts as a cofactor in various enzymes and performs essential roles in photosynthesis, respiration www.nature.com/scientificreports/and the electron transport chain, and is a structural component of defense genes.Excess of Cu, however, imparts negative effects on plant growth and productivity 52 .Irrespective of treatments, growing tuber cowpea raised the soil pH and increased the N, P, K, Fe and Cu contents of soil.Such enhancements may prove to be beneficial for the succeeding crops, with the exception of Fe and Cu which may prove to be toxic at higher concentrations.

Conclusion
The present investigation clearly depicted the effect of plant spacing, blossom retention and deblossoming on nutritional status of tuber cowpea.In principle, the high accumulation of micronutrients viz. the total K, Mg, Fe, Mn, Zn and starch contents was observed in the tubers when flowers were retained on the plants.For developing the tubers with high protein content, the wider spacing (60 cm × 30 cm) with deblossoming can be recommended in zombi pea.In line with this the contents of P, Ca, Zn, Mg, Fe, Cu in green pods and pericarp were also maximum in the wider spacing with inflorescence retention.. Based on the results obtained in studying Zombi pea, we recommend that wider spacing (60 cm × 30 cm) can be adopted for higher protein content in tubers with deblossoming condition.The study revealed that different plant parts of zombi pea comprising pods, pericarp and tubers are enriched with nutrients.Hence, Zombi pea can be a potential commercial crop for combating the malnutrition problem and ill-effects of climate change in the pantropical regions like India and could be a wonder crop in enhancing food security as evident from its nutritional status.

Table 2 .
Plan of layout.

Table 4 .
Analysis of quality parameters of tubers in tuber cowpea.

Table 5 .
Influence of spacing and deblossoming on nutritional value of pod in tuber cowpea.
42sential component of thousands of proteins in plants42.Likewise, the higher accumulation of nutrients like N was reported recently in broccoli under wider spacing byFitsum et al. and Abhijithnaik et al.

Table 6 .
Influence of spacing and deblossoming on protein content (%) of pod in tuber cowpea.

Table 7 .
Influence of spacing and deblossoming on nutrient content of pericarp in tuber cowpea.

Table 8 .
Influence of spacing and deblossoming on protein (%) of pericarp in tuber cowpea.

Table 9 .
Final soil parameters as affected by tuber cowpea at variable spacings and deblossoming treatments.

Table 10 .
Interaction among nutrient components and plant spacing under deblossoming condition.

Table 11 .
Interaction among nutrient components and plant spacing under flower retention condition.