Background 

Mandarin (Citrus reticulate Blanco) is an economically most valuable fruit globally including Nepal (USDA, 2023; Shrestha, 2015; ADS, 2014). Additionally, growing mandarin is very conducive climatically in Nepal as well (Rokaya et al., 2016; NFDC, 2021). Nevertheless, the current productivity is very lower compared to its yield potential (MoALD, 2021). Among the several reasons of low productivity, the declining soil fertility with nutrients deficit condition has been a major problem in Nepal (Prasad and Chandra, 2019). Most of the orchards are rendering under limited use of fertilizers and manures in Nepal (Aryal, 2001). Similarly, the soils of most citrus orchards across the country are reported to have deficit with low level of nitrogen, potassium, calcium, boron, and zinc (Tripathi et al., 2001). Moreover, boron and zinc are the most commonly deficient micronutrients across the citrus growing areas worldwide including Nepal (Srivastava et al., 2001; Tripathi et al., 2001; Alloway, 2008). Eventually, problems of low fruit set, growth and retention exist, which are significant for the higher yield of mandarin. Moreover, heavy fruit drop during different periods is also reported that caused the yield loss to a range of 24 to 76% in Nepal (Subedi et al., 2010; Manandhar et al., 2004; Gautam, 2004; DADO, 2016; Kafle and Rana, 2003). 

Mandarin is a relatively high nutrient demanding crop (Wang et al., 2006) and nitrogen (N), potassium (K), calcium (Ca), boron (B) and zinc (Zn) are the significant to set fruits and yield (Doberman and Fairhurst, 2000; Kaska, 2013). Likewise, there are several reports of the fruit drop due to micro-nutrients deficiency in mandarin (Subedi et al., 2010). In another report (Tariq et al., 2007), micro-nutrients: B, and Zn, including macro-nutrients K, Ca, and N are reported to being critically necessary to boost fruit set and fruit number. So far, information in regard to the response of the plant nutrients on the fruit set, growth and yield of Nepalese mandarin cultivars is very limited. On these contexts, this study conducted to investigate the effects of foliar spray of B, Zn and Ca including N and K on the fruit set, abscission, growth and yield of mandarin in the eastern region of Nepal.

1 Materials and Methods

1.1 Treatments, design and planting materials

The study carried out during two consecutive crop seasons of 2019 and 2020 in the Chungmang Farm of Agricultural Research Station (ARS), Pakhribas, Dhankuta, Nepal (26.22°N, 78.18°E) at an altitude of 1030 meter above sea level. The six treatments: (1) NPK @ 500:250:500 g tree-1; (2) CO(NH2)2 (Urea) @ 1% + KCl @ 2%; (3) NPK + Boric acid @ 0.4% + ZnSO4.7H2O @ 0.2% + CaCl2 @ 1%; (4) Urea @ 1% + KCl @ 2% + Boric acid @ 0.4% + ZnSO₄.7H₂O @ 0.2% + CaCl₂ @ 1%; (5) Boric acid @ 0.4% + ZnSO₄.7H₂O @ 0.2% + CaCl₂ @ 1%; (6) Control were evaluated under RCBD design with four replications; single tree representing one replication. The plant nutrients under the treatments were applied as foliar spray except NPK as soil application. The experiment used local variety, Khoku, of mandarin trees aged of 24 years and planted at a spacing of 5 m x 5 m under hexogonal system in the slightly terrace land. Three foliar sprays were applied: (1) first spray on 27^th February (pre-flowering or 15 days after spring flush); (2) second on 7^th April (through fruit set to early fruit fill); and third on 17^th May (six weeks after second spray). NPK were applied onto the soil in two split parts: 1^st part at last week of January, and 2^nd at last week of May. The spray solutions of Zn, B, Ca, CO (NH2)2 and KCl were prepared in the Soil Laboratory of ARS, Pakhribas using standard procedures. The agro-climate is characterized as sub-tropical, with mild hot summer and cool winter, with mean annual air temperature range of 18 to 34 °C during summer and 4 to 22 °C during winter season. The total annual rainfall was recorded at 1200 ml in 2019 and 1500 ml in 2020 that over 80% of total annual rainfall occurred during June to August. Soil was sandy boulder soil of sandy texture, having low organic matter 1.3%; low NPK and Zn, B and Ca.

1.2 Determination of fruit set, drop, fruit number and yield

Two branches were selected randomly from the middle vertical position of the trees and tagged as the sample branches for the observation. The selected branches size has 63.4 mm, range of 47.8 to 91.3 mm. Additionally, five similar sub-branches from two tagged sample branches of each experimental unit were randomly selected. The bloom fruitlet set was evaluated through counting of fully-developed flowers number, and their set to fruitlets in each two sample branches from 28^th March (full-bloom period) until 1^st May (end of fruit set) at 7 days interval. Likewise, June as well as pre-harvest fruit drop percentage were determined from the same branches in each tree by counting manually the number of fruits retained in the branches, respectively with the references from fruit set 45 days before June and pre-harvest periods on fortnightly basis. Similarly, average fruit diameter and weight (n=10) were measured using digital balance and vernier caliper from the randomly selected sample fruits at the time of commercial harvest. Likewise, total fruit number and yield per tree were measured, and the data were transformed based on the number of fruiting branches of each tree. The fruit set percentage, and fruit drop percentage during June drop and pre-harvest periods were determined using following formula:  

1.3 Statistical analysis

The ADEL-R (Analysis and design of experiments with R for Windows). Version 2.0 was used for the ANOVA analysis; and the treatment mean comparison for significance difference was carried out at 0.05 probability level (Gomez and Gomez, 1984). 

2 Results 

2.1 Post-bloom fruit set 

The percentage of bloom fruit set due to the effects of plant nutrients was studied; since the number of fully developed flowers after post-bloom, and corresponding fruitlets set after petal-fall; were observed for determining the fruit set percentage during bloom period. There were significantly differences (P≤0.01) among the plant nutrients treatments for the total number of flowers and corresponding fruitlets set (Table 1). The bloom fruitlet sets occurred at a range of 2.26 to 5.49%; the maximum fruitlets set (5.49%) was recorded at the combined foliar spray of nitrogen, potassium, and micro-nutrients; closely followed by the combined application of NPK and micro-nutrients (4.13%). Accordingly, these two treatments had 47.9 and 30.7% higher fruit set than that of the control treatment respectively. 

2.2 June and pre-harvest fruit drops 

The fruit drops occurred during June and pre-harvest stages differed significantly (P≤0.05) among the treatments, except pre-harvest drop in 2019 (Table 2). For the June drop in 2019, the treatment of NPK + micro-nutrients had the lowest fruit drop (72.0%) closely followed by NK + micro-nutrients foliar application (72.4%) as compared to the highest fruit drop of 85.6% at the control treatment. Similar results of the respective treatments were observed for the June drop in 2020 since these had the significantly lowest fruit drops, with 16.2 and 15.9% lower fruit drops respectively than the control treatments. For pre-harvest fruit drop in 2019, the lowest fruit drop occurred at foliar application of N + K (22.9%) followed by NPK + micro-nutrients (23.3%), and NK + micro-nutrients (23.9%), compared to the highest fruit drop of 31.1% occurred at the control treatment. Likewise, similar results of pre-harvest fruit drops were obtained for the respective treatments in 2020; accordingly, the lowest drop occurred at NPK + micro-nutrients (23.3%) followed by micro-nutrients (24.5%) among the treatments as 37.2% of the control treatment. 

2.3 Fruit yield characteristics

In 2019, the highest fruit diameter (58.9 mm) and weight (63.4 g) were recorded at the treatment of NPK + micro-nutrients; likewise, the corresponding the highest number of fruits (1790 nos) and total fruit yield (101.81 kg) per tree were also observed at the same treatment (Table 3). The second-best results for the higher number of fruits and yield were observed at the treatment of foliar application of NK + micro-nutrients. Eventually, NPK + micro-nutrients yielded higher fruit number and yield per tree respectively by 124.0 and 102.3% compared to that of the control treatments; similarly, treatment of NK + micro-nutrients had higher fruit number and yield by 93.2 and 66.9% than the control treatment. Likewise, similar results of the corresponding treatments were also observed in 2020 (Table 3). The fruit weight, number of fruits and fruit yield per tree were significantly varied among the treatments. The significantly highest fruit diameter of 44.9 mm, and respective weight of 52.7 g recorded at NK + micro-nutrients; while the highest number of fruits (953 nos) and yield (49.09 kg) per tree were observed at NPK + micro-nutrients. 

3 Discussion 

3.1 Post-bloom fruitlets set 

This study showed the combined foliar application of boron, zinc and calcium with nitrogen and potassium as effective for the improved fruit set by 47.9% higher over the control treatment that the maximum of 5.49% fruit set was achieved. In a study in Florida, Davies (1980) reported final fruit set less than 1% of the total flowers during bloom in navel oranges. Therefore, their effect has significant on the bloom fruit set. Specifically, the initial fruit set during developing fruitlets largely depends on photo-assimilates, and carbohydrate availability (Domingo et al., 2006); hence photosynthesis and photo-synthase production appears to be crucial in developing fruitlets (Mehouachi et al., 2000); as result plant nutrients are the limiting factors. Accordingly, the findings of the study suggest that foliar application of micro-nutrients including nitrogen, and potassium increase photosynthesis rate, eventually resulting in higher fruit set. Similar results of enhanced fruit set by 10% to 25% were reported in mandarin through the foliar application of N-P-K sprays during early and post-bloom since most citrus species requires more K and N for the reproductive growth and fruit set (Albrigo, 2002; Boman, 2001). Moreover, Agustí and Primo-Millo (2020) reported that foliar spray of urea increased the number of leaves that associated with the increase in the biosynthesis of polyamine due to increased ammonium content. Similarly, Embleton et al. (1973) showed that developing fruitlets need nitrogen for protein biosynthesis since its deficiency leads to an intense fruitlet abscission. 

In the study, the possible causes of the treatment for the higher fruit set are the biological functions of nitrogen as associated with photosynthesis and plant growth, causes fruit set and yield (Huang et al., 2021; Liu et al., 2019); while potassium is vital for regulating CO₂ uptake, thus enhancing photosynthesis; and activation of important biochemical enzymes, thus influencing flowering and fruiting. Moreover, the function of boron is associated with the metabolism of nucleic acid, carbohydrates and proteins, especially for reproductive development, is linked to pollination and fertilization of flowers, causing fruit set and growth (Marschner, 2012). Likewise, zinc is an essential component of many enzymes, required for nucleic acid metabolism, and protein and carbohydrate biosynthesis (Srivastava and Singh, 2004); thus, it is vital for new growth of leaves, flower buds, and fruit growth. Similarly, calcium is an important constituent of cell walls, involving in cell membrane integrity (Chaplin and Westwood, 2012); besides an important role in the pollen germination and growth, it also delays senescence and abscission; acting as protein-pectin cement in the middle lamella.

3.2 June and pre-harvest fruit drops 

Domingo et al. (2007) reported over 98% abscission of developing fruitlets during June drop period as it is largely regulated by genetic, metabolic and environmental factors; and fruit growth and retention is supported by the availability of carbohydrates, and mineral elements. In the present study, the treatments: NPK + micro-nutrients and NK + micro-nutrients had significant effect on reducing the fruit drop by 25.1 and 23.2% compared to that of the control treatments respectively (Table 2). Similar result of maximum fruit retention (75.67%) was observed at 2,4-D 10 ppm + Borax 0.5% in Kinnow mandarin (Bagri et al., 2021). The significant effect of micro-nutrients especially zinc and boron is crucial as zinc involves in the synthesis of tryptophan (Gurjar et al., 2018), a precursor of auxin synthesis, resulting in improving fruit retention and growth; additionally, zinc being cofactor of many enzymes affects photosynthesis, nucleic acid metabolism, and protein biosynthesis; therefore, affecting fruitlets growth and retention (Alloway, 2008). Likewise, boron increases pollen germination and pollen tube elongation, leading to increased fruit set; regulates polar auxins transport. Under boron stress condition, it results in excessive fruit abortion and abscission. Moreover, zinc and boron respectively involve in nitrogen and carbohydrate metabolism. Moreover, zinc prevents abscisic layer formation by increased IAA biosynthesis, resulting in decreased of pre-harvest fruit drop. In addition, the synergistic effect of the foliar application of Boric acid @0.2% + ZnSO4 @ 0.5% with gibberellins at fruit set stage was achieved for the maximum fruit set (71.7%) and fruit yield in sweet orange (Khan et al., 2012; Gurjar et al., 2018). Calcium helps in translocating of carbohydrates, and also as being a main component of cell wall as calcium pectate; stimulates lignin and cellulose in abscission layer, resulting in reduced fruit abscission. In the previous study, Talon and Zeevaart (1992) suggested that the reduction in fruit drop may be attributed to the increase level of auxins along with phenolics. Saleem et al. (2005) found the least fruit drop under foliar spray of urea before and after bloom in mandarin. Similarly, potassium as involved in cell division; synthesis of protein, transport of sugar and photo-assimilates into sink (Liu et al., 2000); the improved fruit growth, and reducing drop in mandarin through foliar application of potassium is reported (Lester, 2010; Nasir, 2016). Similar findings of foliar application with K+ Zn were also reported for improving the fruit retention in Kinnow mandarin (Razaac et al., 2013; Saleem, 2008). 

3.3 Fruit yield characteristics 

In the present study, the highest fruit diameter (44.9 mm) and weight (52.7g) were found at the treatment NPK + micro-nutrients among the treatments; likewise, the corresponding the highest number of fruits (953 nos) and total fruit yield (49.09 kg) per tree were also observed at the same treatment. The NK + micro-nutrients performed as the second-best treatment for the higher number of fruits (598 nos) and yield (47.69 kg) as compared to the fruit number (490 nos) and fruit yield (23.75 kg) of the control treatment (Table 3). Similar finding was also reported by Tariq et al. (2007) that the maximum yield of 123.3 kg/tree at Zn + Mn application in sweet orange.  

4 Conclusion

The effect of combined foliar application of N, K, B, Zn and Ca during spring flush period (pre-bloom) had the best result for the fruit set during bloom stage. Likewise, the least fruit drop occurred at the two treatments of foliar sprays: NPK + B + Zn + Ca; and NK + B + Zn + Ca in both June as well as pre-harvest periods. Likewise, the corresponding the highest number of fruits and total fruit yield were also observed at the same treatment. In overall, foliar applications of micro-nutrients viz. boron and zinc including calcium combined with NPK soil application as well as NK foliar spray have resulted as the best for fruit set, and yield of mandarin.

Authors’ contributions

The research design was carried out in collaboration of all authors; ABP principally conducted the field research and data observation including analysis and write of first draft of the manuscript. AKS, KMT and DRB guided experiment execution, data analysis, and manuscript write up. All authors read and approved the final manuscript.

Acknowledgement 

Authors would like to acknowledge Dr Yuba Raj Pandey, former executive director of NARC for granting the fellowship under Global Agriculture and Food Security Fund. Our special thank goes to Mr Phatta Bahadur Baruwal and on-job training students for their help in executing the field experiments.

References

ADS, 2017, Agriculture Development Strategy (ADS) 2015 to 2035, Part I. Government of Nepal, Ministry of Agricultural Development, Singhdurbar, Kathmandu

https://doi.org/10.1016/B978-0-12-812163-4.00011-5

Agusti M., and Primo-Millo E., 2020, Chapter-11 Flowering and fruit set. Editor(s): Manuel Talon, Marco Caruso, Fred G. Gmitter. The Genus Citrus, Woodhead Publishing. 219-244

Alloway B.J., 2008, Zinc in soils and crop nutrition

Aryal K.P., 2001, Optimization of Farm Plan under Mandarin Orange based Farming System in the Western Hills of Nepal. Master Dissertation. Institute of Agriculture and Animal Science, Rampur, Chitwan, Nepal

Bagri U.K., Singh R.S., Verma M., Nagar V., and Yadav P.K., 2021, Effect of Plant Growth Regulators and Micronutrients on Fruit Growth Characters of Kinnow Mandarin (Citrus reticulata Blanco). Int.J.Curr.Microbiol.App.Sci. 10(04): 198-211

https://doi.org/10.20546/ijcmas.2021.1004.019

Chaplin M.H., and Westwood M.N., 1980, Relationship of nutritional factors to fruit set, Journal of Plant Nutrition. 2(4): 477-505

https://doi.org/10.1080/01904168009362791

DADO., 2016, Annural Progress Report (2072/73). District Agricultural Development Office (DADO), Syangja, Nepal

Doberman A., and Fairhurst T., 2002, Rice Nutrients disorder and nutrients management, Potash and Phosphorus. Institute of Canada and International Research Institute, Los Baffios, Phillipines

Domingo J.I., Cercós M., Colmenero-Flores J.M., Naranjo M.A., Ríos G., Carrera E., Ruiz-Rivero O., Lliso I., Morillon1 R., Tadeo F.R., and Talon M., 2007, Physiology of citrus fruiting. Braz. J. Plant Physiol. 19(4): 333-362

https://doi.org/10.1590/S1677-04202007000400006

Domingo J.I., Tadeo F.R., Primo-Millo E., and Talon M., 2006, Carbohydrate and ethylene levels related to fruitlet drop through abscission zone A in citrus. Trees 20: 348-355

https://doi.org/10.1007/s00468-005-0047-x

Embleton T.W., Jones W.W., Labanauskas C.K., and Reuther W., 1973, In: Reuther, W. (Ed.), The Citrus Industry. University of California Press. Vol. 3: 183-210

Gautam I.P., 2004, A Review on Management of Fruit Drop in Mango and Citrus. 2^nd SAS Convention, Agricultural Research for Enhancing Livelihood of Nepalese People. 30 (28)

Gene Albrigo L., and Galán Saúco V., 2002, August, Flower bud induction, flowering and fruit-set of some tropical and subtropical fruit tree crops with special reference to citrus. In XXVI International Horticultural Congress: Citrus and Other Subtropical and Tropical Fruit Crops: Issues, Advances and 632 (pp. 81-90)

https://doi.org/10.17660/ActaHortic.2004.632.10

Gomez K.A., and Gomez A.A., 1984, Statistical procedures for agricultural research. John wiley & sons

Gurjar M.K., Kaushik R.A., Rathore R.S., and Sarolia D.K., 2018, Growth, yield and fruit quality of Kinnow mandarin as affected through foliar application of zinc and boron. Indian Journal of Horticulture. 75(1): 141-144

https://doi.org/10.5958/0974-0112.2018.00025.7

Kafle D.R., and Rana D.B., 2003, Present Status and Existing Problems of Citrus Fruits in Gorkha District. District Agriculture Development Office, Gorkha

Kaska N., 1989, Cell Separation in Plants. Department of Horticulture, Faculty of Agriculture, University of Cukurova Adana, Turkey, NATO AS Series. Vol. H 35

Khan A.W., Malik S., Rashid U., Saleem A.U., and Rajwana I.A., 2012, Exogenous applications of boron and zinc influence leaf nutrient status, tree growth and fruit quality of Feutrell's Early (Citrus reticulata Blanco). Pakistan J. Agri. Sci. 49 (2): 113-119

Lester G.E., Jifon J.L., and Makus D.J., 2010, Impact of potassium nutrition on food quality of fruits and vegetables: A condensed and concise review of the literature. Better Crops. 94(1): 18-21

Liu K., Fu H., Bei Q., and Luan S., 2000, Inward potassium channel in guard cells as a target for polyamine regulation of stomatal movements. Plant Physiology. 124(3): 1315-1326

https://doi.org/10.1104/pp.124.3.1315
PMid:11080307 PMCid:PMC59229

Manandhar R., Sharma P.N., and Devkota S.R., 2004, Monitoring Field Status of Naturally Occurring Egg Parasitoids of Citrus Green Stink Bug (Rhynchocoris Humeralis Thunberg) and Their Identifications. 2^nd SAS Convention, Agricultural Research for Enhancing Livelihood of Nepalese People. 30: 174 

Marschner H., 2012, Mineral nutrition of higher plants. Academic press

Mehouachi J., Iglesias D.J., Tadeo F.R., Agusti M., Primo-Millo E., and Talon M., 2000, The role of leaves in citrus fruitlet abscission: effects on endogenous gibberellin levels and carbohydrate content. The Journal of Horticultural Science and Biotechnology. 75(1): 79-85

https://doi.org/10.1080/14620316.2000.11511204

MoALD, 2021, Statistical Information on Nepalese Agriculture, 2076/77 (2019/20). Government of Nepal, Ministry of Agriculture and Livestock Development, Planning and Cooperation Coordination Division, Statistics and Analysis Section, Singhdurbar, Kathmandu, Nepal. 109

Nasir M., Khan A.S., Basra S.A., and Malik A.U., 2016, Foliar application of moringa leaf extract, potassium and zinc influence yield and fruit quality of ‘Kinnow’mandarin. Scientia Horticulturae. 210: 227-235

https://doi.org/10.1016/j.scienta.2016.07.032

NFDC, 2021, National statistics on fruit crops. National Fruits Development Centre (NFDC), Department of Agriculture, Ministry of Agriculture and Livestock Development, Government of Nepal, Kirtipur, Kathmandu

Panth B.P., and Dhakal S.C., 2019, Determinants of mandarin productivity and causes of citrus decline in Parbat District, Nepal. Acta Scientific Agriculture. 3(10): 14-19 

https://doi.org/10.31080/ASAG.2019.03.0638

Razzaq K., Khan A.S., Malik A.U., Shahid M., and Ullah S., 2013, Foliar application of zinc influences the leaf mineral status, vegetative and reproductive growth, yield and fruit quality of ‘Kinnow’mandarin. Journal of plant nutrition. 36(10): 1479-1495

https://doi.org/10.1080/01904167.2013.785567

Rokaya P.R., Baral D.R., Gautam D.M., Shrestha A.K., and Paudyal K.P., 2016, Effect of altitude and maturity stages on quality attributes of mandarin (Citrus reticulata Blanco). American journal of plant sciences. 7(06): 958

Saleem B.A., Malik A.U., Pervez M.A., Khan A.S., and Khan M.N., 2008, Spring application of growth regulators affects fruit quality of ‘Blood Red’sweet orange. Pak. J. Bot. 40(3): 1013-1023

Saleem B.A., Ziaf K., Farooq M., and Ahmed W., 2005, Fruit set and drop patterns as affected by type and dose of fertilizer application in mandarin cultivars (Citrus reticulata Blanco.). Int. J. Agri. Biol. 7(6): 962-965

Shrestha D., 2015, Production cost and market analysis of Mandarin in Dhading District of Nepal. Journal of Agriculture and Environment. 16: 112-119

https://doi.org/10.3126/aej.v16i0.19844

Subedi H.P., Paudyal K., Srivastava S., Chalise B., Acharya U., 2010, Selection and dissemination of elite genotypes of Acid lime (Citrus aurantifolia Swingle) for off-season production through on farm research in mid and eastern terai regions of Nepal

Talon M., and Zeevaart J.A., 1992, Stem elongation and changes in the levels of gibberellins in shoot tips induced by differential photoperiodic treatments in the long-day plant Silene armeria. Planta. 188: 457-461

https://doi.org/10.1007/BF00197035
PMid:24178375

Tariq M., Sharif M., Shah Z., and Khan R., 2007, Effect of Foliar Application of Micronutrients on the Yield and Quality of Sweet Orange (Citrus sinensis L.). Pakistan Journal of Biological Sciences. 10: 1823-1828

https://doi.org/10.3923/pjbs.2007.1823.1828
PMid:19086544

Tripathi B.P., Shrestha S.P., and Acharya G.P., 2001, Status of micronutrient in the mandarin trees of western hills of Nepal. Lumle Working Paper No. 2001/1, Kaski, Nepal, Agriculture Research Station, Lumle

USDA, 2020, Citrus: World Markets and Trade. United States, Department of Agriculture, Foreign Agricultural Service. https://apps.fas.usda.gov/psdonline/circulars/citrus.pdf

Wang R., Shi X.G., Wei Y.Z., Yang X.E., and Uoti J., 2006, Yield and quality responses of citrus (Citrus reticulate) and tea (Podocarpus fleuryi Hickel.) to compound fertilizers. Journal of Zhejiang university science B, 7: 696-701

https://doi.org/10.1631/jzus.2006.B0696
PMid:16909469 PMCid:PMC1559797