Effect of humic and application at different growth stages of kinnow mandarin ( citrus reticulata blanco ) on the basis of physio-biochemical and reproductive responses

The property of humic acid as a bio-fertilizer in respect of stimulating morphophysiological and biochemical peculiarities of plants is well documented. The present investigation was proposed to delineate the reproductive behavior, physiological attributes and fruit biochemistry of a perennial plant (kinnow mandarin), in response to humic acid. Three doses (40, 60 and 80 ml plant-1) of 8% humic acid (HA), addressed in the drip line of kinnow mandarin plants, with recommended doses of NPK (1000: 500: 500 g plant-1) at three developmental stages viz. before flowering and after fruit setting in two phases, expedited prominent synergistic effects on number of fruits per branch, fruit set per branch and number of fruits per plant. It distinctly contributed towards reduction in fruit drop percentage. Significant positive influence was observed on total chlorophyll contents, stomatal conductance, net photosynthesis rate and transpiration rate. Likewise, a positive effect of HA was detected in cases of total soluble solids, ascorbic acid (vitamin C), total sugars, and reducing and non reducing sugars. However, an attenuating trend was illustrious in case of acidity. Such promising impressions of humic acid can be attributed to stimulation of maximum nutrient uptake by plant and its ability to acquit hormonal behavior. From current investigation, it is screened out that HA application in three splits (before flowering in February, after fruit setting in April and in August), gave fantabulous beneficial response as compared to its single application in a season. Keeping in view the findings of these investigations, humic acid can be cited as an efficient biofertilizer for the improvement of plant performance.


INTRODUCTION
Non-conventional sources of amending organic matter status of soil are acquiring much attention because of their easy availability, prompt response and feasibility in using over large area in less time.Excessive use of inorganic fertilizers creates environment related problems, and situation can be improved through the use of bio-fertilizers (Saadatnia and Riahi, 2009).Humic acid is a valid option as being economical and environment friendly bio-fertilizer.
Use of bio-stimulants and bio-fertilizers like humic acid among these non-conventional sources engrosses prominent position in organic matter deficit soils.Supplementation of these bio-stimulants enhances the fertilizer use efficiency by creating conducive environment for efficacious plant growth (Arancon et al., 2006).
Humic acid is a bio-stimulant, which acts as a growth booster by inflicting positive effects on soil and plant characteristics.It is a complex mixture of aromatic organic acids, with diverse functional groups bearing sulphur, nitrogen, phosphorous, carbon, hydrogen and oxygen in varying percentages and metal ions like calcium, magnesium, copper, zinc etc.It chelates metal ions under alkaline soil conditions and improves the availability of nutrients to plants (Zhang et al., 2010).Moreover, it encourages the conversion of a number of elements like N, P, Fe +3 , Fe +2 (Alva, 1993), Cu, Mg and Pb into available forms to plants (Adani et al., 1998).It heightens the microbial growth in soil and improves the soil physically (Albregts et al., 1988), by stimulating it to be crumbly, modifying aeration (Mackowiak et al., 2001), soil drainage and moisture retention capacity (Salman et al., 2005).
Alkali soluble property of humic acid distinguishes it from other components of humic substances like fulvic acid and humins.It renovates the organic matter status of the soil and increases its suitability to support the vigor and productivity of plant.Humic acid produces dominant effects on plants by stimulating enzyme activity, membrane permeability, photosynthesis (Muscolo et al., 1999), respiration (Nardi et al., 2002), maintaining transpiration rate, increasing protein and vitamin contents (Salman et al., 2005) and yield of dry matter (Liu et al., 1998).
Citrus is widely distributed as a commercial fruit crop (Wu et al., 2011).Kinnow mandarin occupies a discrete position due to its high yield, fresh consumption, aromatic flavor, high processing quality and better adaptation to agro-climatic conditions of Punjab, Pakistan (Ahmad et al., 2006).Imbalance employment of fertilizers and nominal use of bio-fertilizers are the limiting factors, contributing 3 to 4 folds less production as compared to other kinnow growing countries of the world.The production potential of kinnow mandarin can be maximized by encouraging the use of bio-stimulants like humic acid along with synthetic fertilizers, due to its enormous beneficial effects on soil and plant attributes.Humic acid is reported to enhance the vegetative vigor (Eyheraguibel et al., 2008) and reproductive behavior by stimulating the physiological activities, which ultimately contribute towards high production (Karakurt et al., 2009) with superior quality fruits (Mansour and Shabaan, 2007).This research was planned with the objective to optimize the dose and time of application of humic acid and to ascertain the influence of humic acid on reproductive behavior, physiological processes and fruit quality of the kinnow mandarin.

MATERIALS AND METHODS
Humic acid (8%) at concentration of 40, 60 and 80 ml plant - 1 was applied as soil treatment in the drip line of thirty kinnow plants of uniform age (12 years) and size.It was applied at three different growth stages of plant (before flowering, after fruit setting and in August) in single, double and triple splits, considering each as a single treatment.
Cultural practices and recommended doses of fertilizer (NPK and FYM) were applied to all the plants.FYM was applied in December at 40 kg per plant.The data presented is the mean value of two replicates of the complete experiment.

Reproductive attributes
The reproductive behavior of the tree under trial was assessed by tagging 16-branches from each treatment unit.The observations regarding reproductive behavior were made by counting the number of fruits branch -1 , total fruit set branch -1 , fruit drop, and total number of fruit harvested per treatment.Fruit drop percentage on fruit set base was calculated by counting fruits of the tagged branches with a month interval until November.The total fruit drop percentage was calculated by dividing total number of fruit dropped till November by number of total fruit set on same tagged branches which was multiplied by 100.Total yield per tree was determined in terms of number of fruits per plant.

Gas exchange characteristics
Measurements of photosynthesis rate (Pn), stomatal conductance (C) and transpiration (E) were made on a fully youngest leaf of each plant using an open system LCA-4 ADC portable Infrared Gas Analyzer (IRGA) (Analytical Development Company, Hoddesdon, England).Measurements were performed from 9:00 to 11:00 a.m. with the following specifications: molar flow of air per unit leaf area 403.3 mM m -2 s -1 , atmospheric pressure 99.9 kPa, and water vapor pressure into chamber.

Chlorophyll contents
Chlorophyll contents were estimated according to the method by Arnon (1949).Fresh leaves were cut into 0.5 cm segments and extracted overnight with 80% acetone at -40°C.The extract was centrifuged at 14000 × g for 5 min and absorbance of the supernatant was taken at 645 and 663 nm.Total chlorophyll contents were calculated using the following formula: where V is the volume of extract; and W is the weight of the sample.

Biochemical attributes
Total suspended solids (TSS), total titratable acidity, ascorbic acid (vitamin C), total soluble sugars, reducing and non-reducing contents of the fruit were determined, following the methods described by Sattar (1999).

Statistical analysis
The experiment followed the randomized complete block design.The data pertaining to various parameters were analyzed by ANOVA techniques using STATISTICA 8.1 and significance of means was tested using least significant difference at 5% probability (Steel et al., 1997).

Effect of HA on reproductive attributes
Plants that received HA in two splits at 30 ml at two different developmental stages (before flowering and after fruit set), exhibited a significant enhancement in number of fruits branch -1 (93%), while humic acid applied at 26 and 20 ml plant -1 till April exhibited number of fruits branch -1 as 65 and 63% (Figure 1).Higher doses of HA in single application exhibited the minimum percentage of fruit set branch -1 .However, HA applied at concentration of 20 ml till April produced prominent results in case of fruit set branch -1 (83%), followed by 26.6 ml HA (76%) in comparison to control and other treatments (Figure 1).In case of number of fruits plant -1 humic acid addressed at a concentration of 26.6 ml in three splits showed superior performance (39%) as compared to control (Figure 1).Furthermore, plants treated thrice with HA at the same concentration ousted all other treatments by showing maximum reduction in fruit drop percentage by 19%, in comparison to control (Figure 1).

Effect of HA on physiological attributes
Triplicate application of humic acid at the rate of 13.3 ml imposed increment in photosynthesis rates by 97%, being superior to all other treatments in comparison to control.Humic acid applied as a split dose of 26.6 and 20 ml plant -1 caused an increment in photosynthesis rate by 80 and 56%, respectively.However, the humic acid applied in two splits demoed intermediate values of net photosynthesis rate while minimum values were noted when humic acid was applied only once in higher concentration (Figure 1).Maximum increment in stomatal conductance (60%) was calculated in plant acquainted with 20 ml of humic acid in three splits as compared to control, which was close to the percentage increase in stomatal conductance (61%) in plants that received 26.6 ml of humic acid at three developmental stages (Figure 1).On the other hand, minimum values were exhibited by those plants which were addressed by 40, 60 and 80 ml applied in single split.However, plant addressed with 26.6, 13.3 and 20 ml of humic acid in three splits exhibited maximum transpiration rate of 57.23, 55.42 and 45%, respectively, in comparison with control (Figure 2).Single application of humic acid addressed before flowering gave reduced percentage of transpiration rate.Maximum total chlorophyll contents were estimated from plants that received 20 ml of HA (53%) followed by 26.6 ml (45%) and 13.3 ml of HA (34%) in three splits at different developmental stages.The data demonstrated that minimum chlorophyll contents were calculated in the leaves of plants that received 40 ml of HA in a single split before flowering (Figure 2).

Effect of HA on biochemical analysis of fruit
A considerable variation regarding values of total soluble solids, ascorbic acid (vitamin C), total sugars, reducing and non reducing sugars and total titratable acidity among various treatments of HA signifies the profound effect of HA.Maximum increment of 17.84% in case of TSS was found in fruits receiving 20 ml HA at three developmental stages of plants (Figure 2).In contrast, a decreasing trend was observed in case of acidity percentage.Maximum decline in acidity percentage occur by 21.91% in fruits of the plants treated with 20 ml of HA in three splits as compared to control (Figure 2).The highest value of ascorbic acid increment (22.96%) was obvious from the fruits treated with 13.3 ml of HA in three splits as compared to control (Figure 2).Plants which received 26.6 ml of HA in three splits demonstrated highest increase in total sugars by 21.34% as compared to the other HA treatments (Figure 2).However, the trend related to reducing and nonreducing sugars evidenced the prominent position of fruits which received 20 ml in three splits, by an increase of 24.40 and 20.95%, respectively than control (Figure 2).

DISCUSSION
Humic acid has been widely implicated in vegetables (Albayrak and Camas, 2005;Abdel-Mawgoud et al., 2007;Karakurt et al., 2009) and succulent plants, so enormous improvements in vegetative and reproductive physiology of plant genotypes have been reported.However, it has been used in perennial plants to a very limited extent.The activity of humic acid is largely the function of its origin, concentration and pH of the soil solution (Rengrudkij and Partida, 2003).A slightly acidic nature (6.5 to 7.0) of humic acid compels it to lose its functional groups gradually, when applied in alkaline soils.So, it would cause an increment in its availability for a longer period of time to the plant and enhances its ion chelating capability.In the current research, work plants treated with humic acid in split doses exhibited the positive trend in terms of maximum number of fruits per branch, fruit set and number of fruits per plant as compared to single dose application and control (Figure 1).It can be attributed to the strength provided by the humic acid as it has been reported to acquit comparable with auxins (Canellas et al., 2002;Zhang and Erwin, 2004), which have been repeatedly reported to cause a delay in abscission (Taylor and Whitelaw, 2001;Eo and Lee, 2009).
Hence, it can be viewed to induce more fruit set and less flower, fruit and leaf drop.Moreover, HA application increased the ability of plants to maintain higher nitrogen contents along with other micro and macro nutrients (Nikbakht et al., 2008).The enhancement in nitrogen contents caused a significant increment in fruit set so ultimately contributes to maximum yield.The enhanced number of fruits per plant in present study is consequence of enhancement in fruit set, which ascertains the yield.Humic acid increases the number of flowers per plant and the highest flower bud differentiation rate which ultimately boost up the average number of fruits per plant as supported by Arancon et al. (2003) and Yildrim (2007), in case of strawberries and tomato, respectively.Despite other factors, improvement in yield can also be attributed to reduced fruit drop and extension of stay period of fruits on the plant till harvesting.Humic acid plays a vital role in this context by minimizing the drop of precious fruit, through improving the beneficial nutrients (phosphorus and nitrogen) availability to plant and directly preventing the plant from nutritional deficiencies and disorders.It is evident that humic acid application in three splits showed excellent performance to minimize the fruit drop as compared to single application (Figure 1).Presented data signifies that humic acid application in three splits gave the fantabulous performance in terms of maximum stomatal conductance, net photosynthesis, transpiration rate, and chlorophyll contents.It can rightly be said that that HA applied in August strengthen the plants by inducing an enhancement in physiological responses of the plants.Stomata are very important structures within the plant, entailed for gaseous exchange.Their ability to exchange the gases is known as stomatal conductance, which is a leading factor in tempting other physiological processes like photosynthesis and transpiration.Stomatal movements are reported to be regulated by potassium (Taiz and Zeiger, 2006).As humic acid enhanced the nutrient availability to the plant especially N, P and K, so it can also be regarded as a reason for efficacious regulation of stomatal conductance, which thus caused an increment in absorption efficiency of the plant and more efficient exchange of gases along with conversion of raw materials into food.Increased stomatal conductance is directly correlated with higher net photosynthesis and transpiration rate, so HA is regarded as photosynthtic enhancer (Liu et al., 1998;Neri et al., 2002;Muscolo et al., 2007).
The results in this document are in conformity with the findings of Lopez (1993) who reported significant role of humic acid in enhancement of stomatal conductance in apple.Although increase in transpiration rate is not beneficial for plant but stomatal conductance, photosynthesis rate and transpiration rates are interlinked with each other.As HA increased the exchange of gases, rate of transpiration of water from leaf surface also increased because more water is absorbed by roots and more is transpired from leaf surface (Sun et al., 2004).Split application of humic acid stimulated the photosynthetic pigments (Neri et al., 2002), thus aiding in higher photosynthesis rate and efficient plant growth.Higher chlorophyll contents are evidenced from the data, as a consequence of humic acid application in three splits, addressed at different plant growth stages (Figure 2).HA treated plants were apparently distinct from the control plants due to their dark green color and biomass production due to the enhancement in chlorophyll contents.Higher photosynthesis rate is a function of maintenance of high chlorophyll contents (Tejada and Gonzalez, 2003) in leaves.Karakurt et al. (2009) also supported these findings by reporting higher chlorophyll contents in peppers in response to humic acid application.The results related to biochemical attributes of kinnow mandarin clearly manifested the importance of split application of HA, especially the third application in August.Humic acid applied in the month of August positively influenced the fruit quality parameters.As humic acid is recognized as an enhancer of photosynthesis (Liu et al., 1998), consequently there will be more production of assimilates.These assimilates are depicted in terms of total soluble solids (Abdel-Mawgoud et al., 2007).These results are supported by the findings of Zachariakis et al. (2001) and Fallahi et al. (2006) who reported that organic fertilizers containing the highest ratios of humic acid enhanced the TSS in grapes and apple, respectively.The findings of Yildrim (2007) also confirmed these results, according to which, both foliar and soil application of HA enhanced the TSS in tomato.Increased ascorbic acid contents can be justified by the role of HA as nutrient availability enhancer.The HA improves the availability of phosphorous and potassium contents.It is well documented that increased P and K contents increase the ascorbic acid percentage of the fruit (Reuther, 1973).These findings are also verified by Carvajal et al. (1995) and Yildrim (2007).Positive correlation obviously exists between total soluble sugars, reducing and non reducing sugars and split application of HA (Figure 2).The increase in sugars in response to HA might be due to formation of maximum amount of different forms of carbohydrates within the leaf and fruit tissues, which are then converted to the specific sugars like glucose and sucrose.These findings are in confirmation with the findings of Zachariakis et al. (2001) and Zaghloul et al. (2009), who reported enhancement in sugar contents of grapes and Thuja orientalis, respectively.Karakurt et al. (2009) also reported an enhancement in reducing and non reducing sugars in pepper when treated with humic acid.The decreasing trend of acidity percentage can be associated with increase in phosphorous contents (Reuther, 1973;Rajput and Haribabu, 1985).Humic acid proved to be an efficient bio-fertilizer, which improved the nutrient utilization by plant and thus imparted the beneficial influences on plant growth and fruit quality parameters.

Conclusion
Humic acid is emerging as the most prominent bio-fertilizer in enhancing morpho-physiological and biochemical aspects of plant growth.Split application of humic acid at three different growth phases of the kinnow mandarin stimulated the reproductive vigor and physio-biochemical attributes, thus proved to be beneficial for substantiating high yields.