extfieldsofstudy:
Environmental Science
Medicine

text:
Interception Systems in Assessment of Dermal Exposure to Pesticides: Laboratory Comparison of Media

Dermal exposure of workers to pesticides can be assessed using patches, placed on the workers’ clothes or used to wipe off the substance from the skin. Since there are no official indications of the materials to be used for patch sampling, a wide range of materials is suggested in the scientific literature. This paper reports a laboratory study on the affinity of four different pesticides widely used in southern Italy with five patch matrices. Imidacloprid, Hexythiazox, Boscalid and Myclobutanil were tested with cotton and gauze sheets, polyethylene tissue and two different grades of cellulose papers. An aerosol machine was used to nebulize the substance on the patches in a closed system, simulating the conditions of use on the workers’ clothes. The recovery of the analytes from the media was evaluated, by spiking the patches with a known amount of each active substance and testing their performances as skin wipes. Samples were extracted and analyzed in gas chromatography with an electron capture detector. The recovery from the spiked patches was 89–96% for all pesticides, while the test recoveries were very different. Results showed a higher affinity with Imidacloprid and Hexythiazox for gauze, with Myclobutanil for cotton and with Boscalid for paper filters (W41).

Introduction
Pesticides are widely used in modern agriculture to increase the yields of production. Because of their high biological activity, and, in some cases, long persistence in the environment, pesticides may cause harmful effects to human health and to the environment [1]. Occupational exposure typically occurs in workers involved in the manufacture of pesticides and in the agricultural sector, among farmers and professional applicators of pesticides [2]. In recent years, the assessment of occupational exposure to chemicals via the dermal route has grown in importance, in particular for agricultural workers. The current understanding of dermal exposure and uptake has come from researchers investigating the health effects of pesticides [3].
Contact with pesticides can occur the during mixing and loading of products, the application of the spray mixtures and the cleaning of the equipment. Dermal absorption is an important route of exposure in these activities, especially in the mixing and loading, because here the concentrated products are used. Dermal exposure is also significant in closed systems, such as the agricultural greenhouse, where dangerous accumulations of contaminants could occur. The degree of hazard depends on the toxicity of the pesticide, time of exposure and pesticide formulation: powders, dusts and granular pesticides are not absorbed as easily through the skin as liquid formulations, and formulations containing organic solvents and oil-based pesticides are usually absorbed more quickly. Further, the body part contaminated has its role, as certain body areas are more prone to absorption than other areas [4].
Exposure depends also on how the spillage occurs, and on the spraying equipment used. Hand spraying with wide-area spray nozzles is associated with greater exposure for the operators than narrowly focused spray nozzles. Pesticide deposition on different parts of the operator's body depends also on the individual work habits; the hands and the forearms experience the greatest contamination during the preparation and application of pesticides. Re-entry farm workers may be more significantly exposed than applicators, because of lower risk perception, less training on the use of personal protective equipment (PPE), and greater duration of exposure [2]. Occupational exposure becomes significant in closed spaces such as greenhouses, where the high temperature and relative humidity can increase skin absorption [5].
Because of the many possible variables that can influence dermal absorption and the efficiency of sampling methods, the quantitative assessment of dermal exposure is a complex and challenging task [6]. International organizations [7,8] and the scientific literature [9][10][11] indicate different approaches to the quantitative assessment of this type of exposure.
Direct methods include surrogate skin and patch methods, using whole body suits or representative patches to capture the potential exposure, and fluorescence visualization of materials deposited or retained on the skin surface. Washing, wiping or skin stripping techniques can be used to indirectly determine the amount of material present on the skin at a given time point, and biomonitoring to provide an estimate of the amount of actual internal exposure.
Both patch methods and skin wiping involve the use of materials that selectively capture the considered substance and then release it to be analyzed appropriately. The assessment of potential dermal exposure is performed by applying the patches over the clothes, while the effective one is estimated by applying the patches directly onto the skin, under the clothes of the operators, or removing the active substances deposited on the skin. The quantity of a pesticide on a patch with a known area is related to the area of the limb or other body part using standard body part surface areas. Individual body part exposure values are then summed up to give a total potential dermal exposure, that can be expressed in mg/h, mg/d or mg/kg of product handled or applied.
Patches usually consist of two layers, the sampling one and a second one made of an impervious material, such as aluminium or plastic foil, to reduce the potential contamination of the skin, or a loss of pesticide through the patch. The typical thickness of the patches is approximately 1 mm, and the most commonly used size is 10 × 10 cm 2 . They are applied to the worker's body immediately before they start working, and are removed after the end of the working activity. A wide range of materials are used for the scope. Durham and Wolfe [12] published the first review paper detailing methods for measuring dermal exposure to pesticides. Some authors and institutions recommend α-cellulose paper, whereas others suggest alternative materials [13,14], such as cotton, or both polyester and cotton [15]. Filter paper was used for Imidacloprid dermal exposure evaluation [16]. The UK Health and Safety Executive (HSE) [17] recommends using several different patch materials (fabric, polymer, paper, charcoal cloth or composite materials) instead of one material for all substances. It was shown that different substances could have different recovery efficiencies with the same material [18]. Temperature, humidity and the chemical-physical characteristics of substances are variables that must be considered, because they could influence the sampling performances.
The aim of this work was to evaluate the most suitable matrices for performing potential dermal exposure evaluation (PDE), during the application of four chlorinated pesticides widely used in the countryside area of Lamezia Terme. Imidacloprid, Hexythiazox, Boscalid and Myclobutanil are active substances (a.s.) intensively used in the working environments typical of southern Italy, and many agricultural workers are exposed to them on a daily basis. Normally, an aqueous mixture is prepared, to be used with the manual lance or with air-blaster sprayers, depending on the kind of crop or on the workplace area. This paper describes a laboratory study that evaluates the affinity of five of the most-used patch matrices (cotton and gauze sheets, polyethylene tissue and two different grades of cellulose paper) with the four above mentioned organochlorine pesticides.
Imidacloprid is a chlorinated compound, structurally classified as a chloronicotinyl nitroguanidine [19], a novel class of pesticides chemically related to nicotine, which binds to insect cholinergic receptors, causing death at sufficient concentrations [20], and is selectively toxic to arthropods and relatively non-toxic to vertebrates. Under WHO [21] classification, Imidacloprid is in Class II; moderately hazardous. U.S. Environmental Protection Agency (EPA) [22] has classified Imidacloprid into Group E, 'evidence of non-carcinogenicity for humans'. The European Food Safety Authority (EFSA) [23] has delivered its scientific opinion at the request of the European Commission on Imidacloprid, concluding that some levels of acceptable exposure to Imidacloprid might not be protective enough against developmental neurotoxicity. Some cases of poisoning have been also described in the literature [24]. In 2018, the European Commission banned the outdoor use of Imidacloprid because of potential damage to bees, and its use is only allowed in greenhouses [25].
Hexythiazox is an acaricide that acts against the egg, larval and nymph stages. It is classified in group C of U.S. EPA Cancer Classification: 'possible human carcinogen' [26]. The human toxicity excerpts are irritation of eyes, nose, throat and skin.
Boscalid belongs to the group of aromatic heteromonocyclic compounds, a fungicide active against a broad range of fungal pathogens. EPA [27] classified Boscalid with "suggestive evidence of carcinogenicity, but not sufficient to assess human carcinogenic potential", and therefore the quantification of human cancer risk is not mentioned.
Myclobutanil is a triazole chemical used as a fungicide. It is stable under recommended storage and normal use conditions, but can decompose at elevated temperatures. Generation of gas during decomposition can cause pressure build-ups in closed systems. Decomposition products depend on temperature, air supply and the presence of other materials, but can include carbon monoxide, carbon dioxide, hydrogen chloride, hydrogen cyanide and nitrogen oxides [28]. The primary target organ following exposure to Myclobutanil is the liver. Skin irritation and/or gross and microscopic changes of the treated skin were observed after application of Myclobutanil formulations at 100 mg/kg bw/day. The NOAEL (no observed adverse effect level) for local effects is 10 mg/kg bw/day, whereas for systemic toxic effects it is 100 mg/kg bw/day [29].
The compounds studied have also been associated with health problems due to cutaneous exposure [30]. Given their very low volatility, the amount of material inhaled is likely to be scarce, while the dermal exposure can be significant. In assessing dermal exposure, sampling is the first and the most important step, followed by sample extraction and analysis.

Materials and Methods
All reagents were of analytical grade. The active substances of the selected pesticides were PESTANAL grade, purchased from Sigma Aldrich (Milan, Italy). All solvents used for sample processing and analysis were of GC grade. SPE cartridges Supelclean TM columns LC18 (500 mg, 6 mL) were purchased from Sigma Aldrich (Milan, Italy). Calibration standards have been prepared by serial dilution from stock solutions in acetone. The mixtures used for the real use were water solutions in the case of Hexythiazox and Imidacloprid, and aqueous suspensions in the case of Boscalid and Myclobutanil, as a concentration higher than the water solubility of the active substances was used. The pure standards were dissolved or suspended in water in order to reproduce these mixtures (Mixture concentrations are reported in Table 1, together with other properties of the active substances). Temperature and humidity were monitored with a portable hygrometer (Thermo Hygrometer RS1360A, RS PRO Sesto San Giovanni MI, Italy).

Patches Preparation
The patches were made by cutting large sheets into 10.2 cm × 10.2 cm squares, which were used alone for the wiping recovery test, and fixed to a protective backing of aluminium foil, as suggested by the U.S. EPA Residential Exposure Test Guidelines [31] for the aerosol exposure test (Figure 1). cotton wipes (TexWipe ® Cotton TX304) 4.
polyethylene tissue, a synthetic material used to produce protective overalls (Tyvex ® Du Pont) A single sheet of white filter paper was used for each cellulose patch; the same was done for cotton and polyethylene sheets, while for the gauze, three sheets of the same size were overlapped to reach the desired thickness (1 mm).

Analytical Method
The four pesticides were determined by means of a Gas Chromatograph AutoSystem XL GC equipped with an Electron Capture Detector (ECD/GC) (Perkin Elmer Instruments, Waltham, Massachusetts, USA). The capillary column was a Perkin-Elmer 1701, 30 m × 0.32 mm × 0.25 µm. Carrier gas was Helium, at a flow rate of 1.5 mL/min. Split injection mode and a temperature gradient were used, from 150 • C to 240 • C, 2 • C/min, and then to 280 • C, 10 • C/min.
The validation of the analytical method was carried out in accordance with the standard of the Italian body for unification of chemical methods, UNICHIM 179/1 "Guidelines for the validation of analytical methods in chemical laboratories", in accordance with the technical standard UNI CEI EN ISO / IEC 17025: 2005 (General requirements for the competence of testing and calibration laboratories). Single calibration solutions were prepared at four concentration points, ranging from 0.2 to 100 µg/mL, by diluting the commercially available solution of the analyte of interest. Perkin Elmer Totalchrom Software was used for the integration of the areas of the chromatographic peaks generated by the analysis of the calibration standards, to generate a calibration curve. The limit of detection (LOD) and the limit of quantification (LOQ) were determined for each substance on the calibration curve according to the formulas LOD = 3.3 (Sy/S) and LOQ = 10 (Sy/S), where Sy is the standard deviation of the y-intercepts of regression lines and S is the slope of the calibration curve.

Experimental
All experiments were performed in the same laboratory, under a chemical hood, far from direct sunlight and from any sources of heat. The monitored temperatures and humidity were in the range 25-27 • C and 50-60% respectively.
The direct recoveries of Imidacloprid, Hexythiazox, Myclobutanil and Boscalid from the different matrices were determined by adding 10 mL of an ultrapure water solution/suspension of each a.s., at the concentration of 50 µg/mL, to each matrix patch, which was left to dry and then sonicated for 10 min with 10 mL of acetone, purified on a SPE column, and analyzed by ECD/GC. Two replicates were performed for each test.
The standard pesticide solutions/suspensions were prepared in ultrapure water, at concentrations selected to simulate the aqueous mixture of pesticide applied during the following real field applications, according to the product labels: Myclobutanil for the treatment of zucchini, Imidacloprid for flowers in pot, Boscalid for strawberries and Hexythiazox for the citrus fruit.
Two field simulation experiments were conducted for each a.s. A total of 10 filters were used for each experiment, 2 replicates for each one of the 5 different matrices, for a total of 80 filters (4 replicates for each material/active substance pair).
In each experiment, 10 patches were stuck to the inner walls of a test chamber. The chamber was a sturdy cardboard box of 30,000 cm 3 volume (25 cm × 40 cm × 30 cm). The front face was removable to allow easy removal of contaminated patches and the placement of the new ones, while no patches were placed on the box floor, as this area collects all the solution/suspension falling down after being sprayed into the test chamber ( Figure 2). A pneumatic nebulizer for breathing therapy (LAICA S.p.A. Vicenza, Italy) was used to uniformly spray 10 mL of working solution/suspension of one of the four selected pesticide for each experiment through an orifice placed on a face of the box. The air passage through the solution for nebulizing assured the continuous stirring of the suspension, in order to guarantee its homogeneity.
As soon as each solution was consumed (after about 120 min) and the environment was saturated, the machine was turned off and the patches left to dry. Then, the patches were sonicated for 10 min with 10 mL of acetone, and the extracts were cleaned up on a SPE column. The choice of acetone as unique extraction solvent was justified by the common very high solubility of the a.s. in this solvent, as shown in Table 1.
In order to remove the interfering compounds from the pads used in the field, USEPA suggested a variety of cleaning steps (method 8081B). Solid-phase extraction (SPE) is a widely used sample preparation technique. The cartridge was pre-washed with 10 mL of n-hexane and subsequently the extracts were eluted [32,33]. The eluates were analysed by ECD/GC.

Statistics
The present investigation does not require a proper statistical analysis, but all calculations and graphic representations have been performed using Microsoft Office Excel Plus 2016. Data are presented as mean, and standard deviations and percentages have been calculated.

Results
Direct recovery: the mean recovery of the pesticide from the five different patch matrices ranged between 89% and 95.6% for all pesticides, indicating that all of them can be used to wipe off the substances from the skin ( Table 2). Field exposure simulation experiments were conducted as described above, and completed over a few days, keeping the extracted samples in vials at 4 • C. The samples were treated as indicated in the literature for the assessment of dermal exposure [34]. The amount of active substance for each pad was determined. The mean amount of active substance found for the four replicates, with the relative standard deviation, is reported in Table 3. The recoveries of the active substances can be calculated from the total sprayed amount, assuming a homogeneous distribution of the sprayed mixture inside the chamber, and a normalized recovery can be calculated by assigning the value of 100 to the sum of the recoveries. These values are reported in Table 4. Form the recoveries reported in Table 4, different behaviors are highlighted for each of the five media, and the relative distribution of each pesticide among the different matrices, expressed as the normalized recovery, is visually shown in Figure 3. The results show that all these materials can be used when skin wiping has to be performed, as they only have to mechanically remove the pesticide from the skin and absorb it by contact. If the materials are used to produce pads, to be placed on the workers clothes to passively absorb the pesticide aerosol, the different affinities of the materials with the active substances play an important role, leading to significantly different results. The gauze seems to be the most appropriate material for Imidacloprid and Hexythiazox, while a material that gives acceptable results for all active substances is polyethylene, which is also used for producing working clothes of farmers. Only Myclobutanil shows an affinity with cotton, and Boscalid with one type of paper filter (W41).

Discussion
The materials selected to produce the pads are all readily available. Polyethylene fabric is frequently used as a synthetic material for protective suits (Tyvex brand), while cotton and gauze are often used for gloves. Filter papers are commonly used in chemical laboratories. The patches are made in accordance with recommendations by the Organization for Economic Co-operation and Development (OECD) [16]: that the collection area of each patch be approximately 100 cm 2 in size, as patches smaller than 50 cm 2 are generally inadequate. The same paper also suggests aluminium foil as a backing material.
Results on direct recovery are in agreement with the findings of Aprea [17], who showed about 100% direct recovery validation of filter paper for Imidacloprid.
The analysis of the materials' properties could help in understanding their behaviors towards the selected pesticides. Polyethylene consists of nonpolar, saturated, high molecular-weight hydrocarbons, and therefore, it absorbs almost no water, but can absorb some solvent, oil and plasticizers [35]. This can justify its moderate affinity with all the selected active substances that are organic compounds, having low or very low water solubility. On the other hand, the presence of hydroxyl groups on the cellulose macromolecules imparts hydrophilicity to cotton, gauze and filter papers, which preferably absorb the more water-soluble substances. This is verified for the gauze, which preferably retains Hexythiazox and Imidacloprid. Myclobutanil seems to be well retained by cotton, but it must be remembered that this pesticide has a very poor solubility in water, and it is sprayed as an aqueous suspension, so it could be physically retained by the woven-fibers structure more than because of its chemical affinity. The same hypothesis can be made for filter paper W41, which is a cellulose paper for coarse particles, and which showed good recovery for Boscalid.
In accordance with what is found in the literature [36], where gauze is used to make patches for pesticide evaluation, especially for powders and granules, a good affinity with Imidacloprid and Hexythiazox was shown for this material. As Imidacloprid and Hexythiazox are often mixed together in the same solution, the possibility of using the same material as a sampling media is an advantage.
When the intent is to use a single matrix that fits all four pesticides for dermal evaluation, surely the gauze or polyethylene seem to have better results, while a specific material can be used if only a single substance is monitored.
Our results are in agreement with J. Davis [37], who confirms that the type of material used for construction of cloth patches may have a considerable effect on the amount of toxicant that they will retain.
There are not so many published laboratory studies to compare with our findings, as field exposure assessment studies take into account other variables, like the different parts of the body where the patch is applied, the comparison with inhalation exposure, and the comparison of the potential exposure with the total applied quantity of pesticide, all for the same active substance [38].
This study has some limitations, as the active substances have been used instead of the formulated products, and even if results give important information, the simulation does not match exactly the field operations. The real airborne concentration of each pesticide during spraying is not measured, but this does not affect the quality of results. In fact, this is a comparison study, and even if all substances and interception media pairs are treated the same way, different behaviors are highlighted. Further investigations will be aimed at determining if the relative affinity of the active substances with the used materials would be influenced by the use of the formulated products in the simulation.

Conclusions
Despite the great relevance of dermal exposure assessment for agricultural workers, there are not so many papers studying the affinity of different absorbing media with specific active substances, and this is one of the very few papers examining this important issue.
All the tested media can be used when skin wiping has to be performed. If the materials are used to produce pads, significantly different results are found. The gauze is an appropriate material for Imidacloprid and Hexythiazox, polyethylene gives acceptable results for all active substances, Myclobutanil shows an affinity with cotton, and Boscalid with W41 paper filter.
This study provides preliminary indications of the type of materials to be used for the dermal sampling of Imidacloprid, Hexythiazox, Boscalid and Myclobutanil, and suggests a method for the laboratory simulation of field operations, that can be applied to more active substances and to other sampling media.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

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Physico-chemical, nutritional and functional characterization of Pathernakh pear ( Pyrus pyrifolia L.) from Himachal Pradesh of India

The present study was conducted to evaluate the physico-chemical, nutritional and functional properties of pathernakh pear grown in Himachal Pradesh. The physical properties (weight, length, width and colour), chemical and nutritional properties of the pathernakh pear including moisture, ash, total sugar, reducing sugar and minerals of pathernakh pear were studied. Moreover, the pear variety was characterized for functional components with respect to total phenols, ascorbic acid and fiber analysis. The results indicated that the fruit is a good supplier of sugars, minerals, ascorbic acid and polyphenols. The edible portion of the fruit possess a high level of fiber content. Additionally, textural properties in terms of firmness of the fruit were also studied. The overall results concluded that pathernakh pear could be best for consumption owing to superior nutritional and functional properties that has many health benefits.

Introduction
Fruits are the dense sources of essential nutrients and considered as functional foods. They are the major sources of phytochemicals such as vitamins, carbohydrates, minerals and dietary fiber. Pear fruit is grown in temperate and subtropical environment due to its broad adaptability to environmental conditions. In India, it is grown in semi-temperate regions of states of Himachal Pradesh, Punjab, Haryana, Utter Pradesh and North East region. Pathernakh is a variety of pear that is known so, because of its hard texture. The fruits of Pathernakh are mostly like by consumers owing to its crispness, sweetness, fragrance and unique flavour. It has grit cells that improve its eating quality. The fruits of Pyrus genus have high nutritional and functional value as common pear provides 11.90g of carbohydrates and 52 kcal of energy per 100 g of edible portion. It also possesses health-promoting bioactive compounds such as carotenoids and plant sterols (Andreotti et al., 2006) [2] . Moreover, pears are fat-free and a good source of potassium, all plays an important role in disease prevention (Mariana et al. 2010) [8] . Total sugars, total soluble solids and titratable acidity are the certain chemicals that have great influence on organoleptic properties of the fruit (Teng and Liu, 2009) [13] . Besides the above chemical properties, various functional compounds are also identified in pears (Chaalal et al., 2013) [6] . Pear fruit also contains a high amount of moisture thereby highly perishable and spoil rapidly that result in unexpected economic losses. Henceforth, keeping all these points, the present study was conducted to evaluate the nutritional and functional components of Pathernakh, whether, they are similar to common pear or not. So, that it provides a better understanding for researchers to produce or to think of new products and convenient processing characteristics of the pathernakh fruit in order to preserve it for longer period or maintain it's fresh like characters even after harvesting.

Materials and Methods
The fruits of pathernakh pear (Pyrus pyrifolia) harvested at optimum maturity were procured from the local market of Solan Himachal Pradesh. Fruits were collected randomly and brought to the laboratory for carrying out the present study.

Physical characteristics of pathernakh pear Size
The size of the fruits was analyzed by measuring the length and width with the help of digital vernier caliper. The average fruit size (length and width) was expressed in millimeter (mm).

Weight
The weight of ten fruits was taken by using digital weighing balance individually and then average of all was expressed as fruits' weight in grams (g).

Textural Analysis
Firmness one of the parameter of texture analysis was determined using a TA-XT2i texture analyzer (Stable Micro Systems, Godalming, UK) connected to a computer. Ten fruit were selected randomly for this. A force was applied to produce a 5 percent deformation by a 100mm aluminum probe. The slope was determined in the linear zone of the force deformation curve and the results were expressed in bioyield point and firmness value in gram (g).

Colour analysis (Ranganna, 2009) [12]
Colour of samples was measured in a 'Lovibond Colour Tintometer Model PFX-I series spectro-colorimeter' in which Red, Yellow, Blue and Neutral (RYBN) colour were obtained along with CIE readings i.e. L * , a * and b * values. The L* value gives a measure of the lightness of the sample colour from 100 to 0, 100 for perfect white while 0 for black colour, a* value represents the green to red colour range and b* values indicates yellow to blue colour range. The colour of each sample was measured thrice.
Chemical parameters of pathernakh pear Moisture content (AOAC, 2000) [3] Weighed sample in replication (5.00g) was taken and dried at 70 ±2 0 C in hot air oven to a constant weight. Loss in weight of sample after drying denotes the moisture content and expressed as per cent (%).
(Weight of fresh sample -Weight of dried sample) Moisture (%) X 100 Weight of fresh sample Ash (Ranganna, 2009) [12] Total ash was estimated through gravimetrically by taking sample of 5.0 g in silica crucibles and burnt on a hot plate. Thereafter, crucibles were placed in a muffle furnace for ashing at 550 °C for 5-6 hrs to obtain a carbon free white ash sample with a constant weight. The ash content of samples was calculated and expressed as per cent (%) on fresh weight basis as given below: Weight of ash Ash (%) = X 100 Weight of sample Titratable acidity (AOAC, 2000) [3] Titratable acidity was estimated by titrating a known volume of the sample against standard 0.1 N Sodium hydroxide (NaOH) solution using phenolphthalein indicator till the end point (pink colour). The titratable acidity was expressed as per cent (%) malic acid.

Total soluble solids (Ranganna, 2009) [12]
The Total Soluble Solids (TSS) of samples were determined with the help of hand refractometer of (Model ERMA). [12] For sugar estimation, a known amount of crushed sample (25 g/ml) was taken and dissolved in 100ml water. Solution was neutralized with 1 N sodium hydroxide and 2ml of 45 per cent lead acetate was added to it and kept for 10 min. 2ml of 22 per cent potassium oxalate was added after it and the solution was filtered and clear filtrate was used for estimation of reducing sugars by titrating against Fehling's A and Fehling's B solution using methylene blue indicator (Lane and Eynon, 1923). Reducing sugars were estimated thereafter and calculated as given below: mg of invert sugar x Dilution Reducing sugars (%) =

Sugars (Ranganna, 2009)
x 100 Titre value x Wt. or volume of sample However, total sugars were estimated by addition of 5g of citric acid to 50ml filtrate from the reducing sugar estimation and heating it for 10min, then similarly neutralizing it with 1N NaOH using phenolphthalein indicator and total sugars were estimated as given below: Functional parameter's analysis of pathernakh pear Crude fiber (AOAC, 2000) [3] One gram of sample was taken in crucibles for fiber determination through 'Fiber Tech Analyzer (Velp Scientifica)'. Then, crude fiber was determined by continuously washing with sulphuric acid, hot water and potassium hydroxide (KOH) and again with hot water followed by cold water and acetone solution (25ml). Thereafter, crucibles contained fiber content were dried in oven at 105 ˚C for an hour up-to constant weight. This weight represents crude fiber with ash content (F 1 ). After that, crucibles were placed in muffle furnace and heated up-to 550 ˚C for 3 hour. Reweigh the samples after cooling in dessicator (F 2 ) and crude fiber was calculated by using following formula: Crude fiber (%) = F 1 -F 2 / F 0 × 100

Total phenols (Bray and Thorpe, 1954) [5]
The amounts of total phenols in the sample were determined with the Folin-Ciocalteu Reagent (FCR) using catechol as a standard. One gram of sample was taken and crushed with 10ml of 80 per cent ethanol and centrifuged at 10,000 rpm for 20 min and then filtered. Filtrate was evaporated in oven up to dryness and residue was dissolved in 5ml distilled water. From that, aliquot of 0.2 and 0.4ml was taken in separate test tubes and volume was made up to 3ml with water. After that, 0.5ml Folin-Ciocalteu Reagent (FCR) was added and incubated for 3 min. Twoml of Sodium carbonate was added to it. Test tubes were placed in boiling water bath for one min and then cooled and OD of the sample was recorded at 650 nm.

Ascorbic acid (AOAC, 2000) [3]
The ascorbic acid content was estimated as per AOAC method using 2, 6-dichlorophenol indophenol dye. Results were expressed as mg per 100g of sample and calculated by using the following formula: Titre value × Dye factor × Volume made up Ascorbic acid (mg/100g) =  100 Aliquot of extract taken × Weight of sample taken

Minerals' analysis of pathernakh pear (Rajasekaran et al. 2005) [11]
The dry ashing method was used for mineral quantification. Ashing was done to free all of the samples from the organic materials. Two gram of ash was digested with a mixture of hydrochloric acid (HCl) and nitric acid (NO 3 ) in the ratio of 1:3. The digested samples were dissolved in 50ml of distilled water and used for the estimation of mineral content through atomic absorption spectrophotometer.

Results and Discussion Physico-chemical characteristics
The physico-chemical characteristics of pathernakh pear are presented in Table 1, 2 and 3. The length, breath and weight of fruit were observed 59.00 0.15mm, 58.70 0.10mm and 148.00 4.02g, respectively. The moisture content determines the freshness of fruits and were found 84.950.10 percent. The minerals content expressed as ash content, total soluble solids (TSS), titratable acidity, ascorbic acid and fiber content that determine the fruit quality were recorded 1.90 0.01 percent, 13.00 1.05 0 B, 0.34 0.01 percent, 4.50 0.20 mg/100g and 6.02 0.25 percent in fruits, respectively. Sugar also an important component of fruits that correlated with sweetness, aroma, texture and flavor were reported 8.77 0.14 percent, respectively. Similar findings have been reported by Yim and Nam (2015) [15] in sand pear. However, in pear, total phenolic content (150.33 0.16mg/100g) were observed. The results were in conformity with the findings of Abaci et al. (2015) [1] .

Textural (firmness) analysis
Firmness is an important textural component that describes the mechanical properties.  [14] and Baniwal and Singh (2017) [4] reported the firmness value of 1.61kg and 3.21Nmm -1 in raw pear fruits. This change in firmness value may be owing to varying geographical conditions, maturity stage and varietal difference (Musacchi and Serra, 2017) [9] .

Colour analysis
The color is most critical parameter that defines the acceptability of fresh as well as processed products (Pingret et al. 2013) [10] . The colour values L*, a * and b * of raw pathernakh pear are shown in Table 4. The pear fruits were yellowish green with L* 47.22, a * 2.23 and b * 40.06 respectively.  [7] reported a higher concentration of minerals in pear fruit. The difference in mineral compositions of fruits may be attributed to varying growth conditions, soil properties and geographical environment.

Conclusion
The present study is the evaluation description of the physicochemical, nutritional, textural as well as functional characterization of the pathernakh pear. It was demonstrated that fruit has better physico-chemical properties and high nutritional compounds such as reducing sugars, ascorbic acid, total sugars and various minerals. Also, possess the high fiber content and high amount of total phenolic content. These results cocluded that this, pear cultivar could be best for consumption as fresh or after favourable processing because of excellent product quality and good concentrations of functional and nutritional components.


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Research Progress of Soil Water Infiltration

Soil water is an important link in the transformation of the “five waters”, which determines the growth and production of crops. Mastering the scientific infiltration law is a key link to promote agricultural development. Based on the existing research results, this paper systematically summarizes the basic theories, test methods, influencing factors, simulation equations, prediction models and other results of soil water infiltration research at home and abroad so far, and discusses the future research development direction.

Introduction
Soil water refers to the water in the soil layer below the ground surface and above the groundwater surface (phreatic surface), also known as soil water in the unsaturated zone [1]. The movement of soil water is considered to be the most important part of the hydrological cycle and an important link in the transformation of the "five waters" (precipitation, plant water, surface water, soil water and groundwater). It plays a key role in the material circulation in the process of ecological development [2]. The process of soil infiltration is the link that connects surface water, groundwater and the bio-earth cycle, and affects the replenishment of soil water and the effective degree of soil moisture storage. As the only type of water that can be absorbed and used by plants and an important part of the field soil water cycle, soil water determines the growth and production of crops [3][4][5][6][7]. In the ecosystem, it maintains a dynamic energy balance and Material transmission [8]. All these fully illustrate the importance of soil water in the earth's ecological environment, and also reflect the necessity of soil water infiltration research [9].
Based on the existing research results, this paper systematically summarizes the basic theories, test methods, influencing factors, simulation equations, and prediction models of soil water infiltration research at home and abroad so far. This article analyzes the existing research results and their remaining shortcomings, discusses future research development directions, and provides references for further research on the process of soil moisture infiltration, in the hope that the continuously developing infiltration theory can be better applied in production practice and promote social development.

theory of soil water infiltration
The development of the basic theory of soil water infiltration has experienced the infiltration from one-dimensional vertical infiltration to multi-dimensional infiltration, from indoor artificial environment to outdoor natural test environment, from single variable to multiple variables, from saturated infiltration theory to unsaturated infiltration theory, from the equation model of infiltration process to the prediction model of infiltration parameters, from discipline branch to independent discipline, it has been more than 100 years since its development [10].
In the middle of the eighteenth Century, based on the importance and practical significance of infiltration process, relevant scholars in western countries carried out a series of theoretical studies on soil water infiltration and put forward many important results. In 1856, French engineer H.P.G. Darcy obtained the equation describing the relationship between the seepage velocity of water in saturated soil and the seepage slope through experiments, and this equation is also called the Darcy's law [11].In 1877, Brigs L. J observed the existence of surface tension in the experiment as the main reason for the retention of soil moisture, and then put forward the capillary hypothesis. The development of the theory of soil moisture infiltration began to transition to the micro field [12]. In 1907, Buckingham put forward the capillary potential theory. In 1920, the relationship between soil water potential and water content was obtained by Gardner study, so that the viewpoint of energy began to intervene in the research process of soil water infiltration [13].In 1931, Richards used mathematical methods for the first time in the research and calculation of unsaturated soil water infiltration. The theoretical study of soil infiltration began to develop from the direction of saturated one-dimensional infiltration to unsaturated multi-dimensional infiltration, and in the direction of quantitative and dynamic research [14].In 1957, Philip successfully used the thermodynamic function to express the movement and distribution of soil moisture in the research process. This form has been widely applied [15]. In 1966, Philip proposed a more complete concept of the SPAC system. Through the unified energy index of water potential, he pointed out that the water in the system always moves from a higher water potential to a lower water potential. The study described the energy transfer process between various elements in the system from a quantitative perspective, and pushed the research and development of soil infiltration theory to a new stage [16]. In the 1950s and 1960s, China's theoretical research on soil moisture infiltration began, and it mainly used the morphological research viewpoints and methods of Soviet scholars to carry out applied research [17][18]. In 1988, Lei Zhidong et al. [19] published Soil Dynamics and proposed a solution to the spatial heterogeneity of soil water dynamic changes. In 1996, Zhang Weizhen et al. discussed and summarized the dynamic relationship and analysis methods between groundwater and soil water, and published Groundwater and Soil Hydrodynamics. In 1997, the first national soil physics academic seminar was held, at which the energetics research viewpoints in the process of soil infiltration were summarized and promoted. Since then, Chinese scholars have carried out in-depth research on the measurement and calculation methods of soil water infiltration SPAC system water and heat flux [10].

soil moisture infiltration test method
The research of soil moisture infiltration has been developed so far, and many soil infiltration measurement methods have been produced in experiments. In the early stage, it can be divided into field test and indoor measurement, including water injection method, double loop method, single loop method, artificial rainfall method, hydrology method and time domain reflectometer (TDR) method [20][21]. Due to the different principles and operating methods of different infiltration methods, the measurement results and their accuracy are often different.

irrigation method
The water injection method is the most classic method. This method calculates the infiltration rate by recording the amount of infiltration within a certain period of time. The test usually uses double-ring, constant-head equipment, and keeps the inner and outer rings of the infiltration equipment at the same water depth. This method saves time, effort and water, and is widely used in field flat ground experiments. However, it is limited to single-point test, and it is difficult to simulate the infiltration process and law under different slopes and rainfall intensity [22][23].
The basic principle of the Time Domain Reflectometer (TDR) method is to use probes to measure the time when electromagnetic waves encounter obstacles in the soil, to obtain the dynamic changes of soil moisture content, and then to estimate the soil moisture content. This method can monitor the change of infiltration rate at a fixed point for a long time, and the determination speed is fast and the accuracy is high [24].
Hood infiltration meter is a new type of tension infiltration meter. It conducts water through the water pipe, automatically collects and records the saturated hydraulic conductivity and infiltration rate of the soil, and the readings are relatively accurate and closer to the actual value [25]. Afterwards, the output data can be graphed through the software, and the measurement results are intuitive and clear. However, it still has shortcomings such as small infiltration area and lateral infiltration of instruments, and it cannot describe the dynamic infiltration process of soil water [26]. It needs to be improved in the future.
The Guelph infiltration meter can measure the soil infiltration rate, and its basic principle is similar to that of a Markov flask (constant head). Using the Guelph infiltration meter to conduct indoor tests can obtain infiltration parameters such as soil infiltration amount and wet front, and can observe the dynamic change process of parameters. It is easier to control the test process and can fully express the soil infiltration performance [27].
The disc infiltration meter method is a new method to determine the hydraulic parameters of soil infiltration. It is simple to operate, short in measurement period, and consumes less water for testing. The effect is better than the double loop infiltration meter method. Within a certain range, this method can be used to determine the hydraulic parameters of soil infiltration at any depth above the groundwater level. In addition, the disc infiltration meter can eliminate the influence of soil cracks, earthworm boreholes, plant root holes and other pores on the measurement [28]. This method has been widely used in soil water research and experimentation.

hydrology law
Hydrological method refers to the method of comparing and analyzing the actually measured rainfall or runoff test field data in a small watershed, and then using mathematical statistical analysis methods in hydrology to derive the soil infiltration equation, and then obtain the average soil moisture infiltration rate. The measurement results obtained in this way are more accurate, but the data collection process is more difficult, and the dynamic process of soil moisture infiltration cannot be obtained. It is suitable for use when measuring the infiltration rate in a large range [29-30].

artificial rainfall method
Compared with other methods, the artificial rainfall method can better simulate the situation of natural rainfall, and the water balance method can be used to estimate the amount of soil moisture infiltration. Its advantage is that it is less constrained by terrain such as slope and height, and can control the measurement time, precipitation intensity and other factors. However, it is not suitable for operation in the field, and has the disadvantages of high cost, large size, labor and time-consuming [31].

dynamic monitoring platform for soil moisture infiltration
After the 1990s, with the continuous development and progress of computer science and "3S" technology, countries began to build large-scale long-term soil moisture infiltration dynamic monitoring platforms, and gradually realized large-scale long-span monitoring. However, this technology still has limitations such as low spatial resolution and complicated relationship between acquisition parameters and soil moisture [32].

factors affecting soil water infiltration
According to the existing research results, the main factors affecting soil infiltration can be summarized into two points. On the one hand, soil water infiltration characteristics are mainly determined by the influence of soil bulk density, soil initial water content, soil texture, soil type, soil structure and soil organic matter and other physical and chemical properties of the soil; on the other hand, vegetation coverage, precipitation and Human interference and other factors outside the soil will also have a significant impact on the process of soil moisture infiltration [10].

soil bulk density.
Soil bulk density is the ratio of the weight of a certain volume of soil after drying to the same volume of water weight, and soil infiltration capacity is greatly affected by soil bulk density [33]. Many related studies have found that the soil infiltration rate, cumulative infiltration amount, and distance of soil wet front movement all decrease with the increase of soil bulk density. And the influence of soil bulk density on the infiltration rate gradually weakens with the increase of time [34][35][36]. Research by Li Zhuo et al. [37] showed that the relationship between soil bulk density and the number of macropores and the cumulative infiltration volume at 120 min is very similar, which shows that the influence of soil bulk density on infiltration characteristics is essentially realized by changing the number of macropores.

soil texture.
Soil texture is the combination of soil particles of different diameters in the soil. It is mainly divided into sandy soil, clay, loam, silt and other types according to the composition of soil particles, which has a significant impact on the soil infiltration process. The soil texture changes from light to heavier, the soil infiltration rate decreases, and the infiltration capacity decreases accordingly [38]. There is also a significant correlation between soil texture and infiltration capacity. Soils with tight structures have stronger infiltration capacity than other loosely structured soils. After the loose soil is compacted, its infiltration rate will be reduced to 2% of that before the loose soil compaction [39].

soil contains organic matter .
Soil organic matter generally refers to life-derived substances in the soil, including soil microorganisms, soil animals and their secretions, and plant residues and plant secretions in the soil [40]. Compared with soils lacking organic matter, soils rich in organic matter have changes in soil porosity and adsorption of soil particles, which will have a certain impact on the soil infiltration process. Shan Xiuzhi et al. [41] found that different organic matter content in soil has different influence processes on soil properties. When the organic matter content is in the range of less than 15.0g/kg, under certain other conditions, if the organic matter content increases, the soil saturated hydraulic conductivity will increase. When the organic matter content is greater than 15.0g/kg, under certain other conditions, if the organic matter content increases, the saturated hydraulic conductivity of the soil will decrease instead. Zheng Jian [42] studied the effect of biochar on soil organic matter and nitrogen content and found that adding biochar to the same bulk density soil can accelerate the infiltration rate. Within a certain range, as the mixing thickness and mixing amount of biochar increase, the infiltration rate also increases.

Initial water content of .
Soil water content is the amount of water contained in the soil, which directly affects the initial infiltration rate of soil surface water [43]. With the increase of soil water content, the water absorption of soil particles will cause volume expansion (if the soil particle colloid content is large, this phenomenon will be more obvious), which will reduce soil pores and reduce water absorption, which affects soil water intake. Infiltration process [44]. Jia Zhijun [45] and Zhang Zhiguo [46] obtained the relationship between slope soil infiltration property parameters and soil water content through measured data in experiments, and the results showed that the two are linearly negatively correlated. The higher the initial soil moisture content, the shorter the time for the soil to reach a stable infiltration rate and the lower the infiltration rate. Li Fa wen [47] showed that under certain other conditions, as the initial soil moisture content increases, the cumulative infiltration volume decreases.

surface crust.
Surface crusting refers to the phenomenon that some fine particulate matter fills the pores of the soil surface due to the impact of raindrops and surface runoff during the rainfall process, which leads to the phenomenon that the surface texture of the soil tends to be dense and the bulk density increases [39]. Too dense soil crust will reduce or even completely disappear the soil moisture infiltration channels, which will greatly weaken the soil infiltration capacity. Jia Zhijun et al. [48] found that the soil water content and infiltration capacity of the 0-50cm loess plough layer were significantly affected by the surface crust. After removing soil crusts, it improves soil properties, increases soil water retention capacity, enhances infiltration capacity, and reduces water evaporation.

vegetation coverage.
The vegetation coverage rate reaches a certain level, and it can directly affect the local hydrological and ecological environment by reducing the evapotranspiration of precipitation, redistributing soil precipitation, reducing rainwater into the surface runoff and other methods [49]. According to the research results of Zhang Zhi qiang et al. [50], the succession of different types of vegetation in the subalpine dark coniferous forest area in the upper reaches of the Yangtze River will affect the saturated hydraulic conductivity of the soil. Wang Genxu[51] studies on alpine meadow vegetation also showed that vegetation has a significant impact on soil infiltration. Wang Bang tuan [52] and others have shown that vegetation coverage affects the infiltration process, and vegetation development will accelerate rainwater infiltration. Bai Sheng yuan [53] used loess soil pillars to study the effect of vegetation coverage on infiltration depth and found that different coverage methods and coverage have different effects on enhancing soil infiltration capacity.

precipitation.
At present, the influence of rainfall intensity on the process of soil water infiltration has not yet formed a unified conclusion. Zhao Xi ning [54] and Shi Sheng xin [55] believed that during the precipitation process, the surface of the soil was directly impacted by water droplets, and the natural texture of the surface soil was disturbed, causing part of the originally static capillary pore water to be destroyed and released into the water flow. And the study found that the steady infiltration rate of soil increases with the increase of precipitation intensity. Yang Yonghui [56], Helalia A M [57], etc. believe that small particles carried by raindrops or runoff produced by rainfall can easily block the pores of the surface soil, resulting in skinning and other phenomena. With the increase of precipitation intensity, the greater the impact on the surface soil, the less stable soil infiltration rate. The physical and chemical properties and surface characteristics of the soil will affect the process of soil water infiltration during precipitation, so different conclusions may be drawn for different soil types and external conditions. In the existing related experimental studies, artificial rainfall is mostly used with constant rainfall intensity. However, the influence of precipitation in the actual environment on the process of soil water infiltration is very complicated and has many factors.

simulation model of soil water infiltration process
With the continuous development of soil infiltration theory and experience, scholars have begun to study the simulation model of soil water infiltration to describe the infiltration process of soil water. The following is a widely used soil water infiltration simulation model in practice and experiment.

prediction model of soil water infiltration parameters
Due to the complexity of water movement in the unsaturated zone in the soil and the temporal and spatial variability of natural soil, so it is very difficult to obtain scientific and accurate soil water infiltration parameters through field tests [58]. The soil transfer function method is a method for predicting and obtaining soil water infiltration parameters that are difficult to obtain accurately through basic soil data that is available or relatively easy to obtain [9]. Regarding the method of establishing soil transfer function, the current applications include linear regression analysis model, nonlinear regression analysis model and artificial neural network model.

linear regression prediction model for soil water infiltration parameters
In 1979, Gupta and Larson [59] established a multiple linear regression fitting model for the first time, using basic physical and chemical parameters of soil including texture, organic matter, and density as input parameters to predict soil moisture characteristics and meet the accuracy requirements. In 1999, Minasny et al. [60] established a linear prediction model, using soil texture and density as input factors to predict soil moisture content. At the same time, it is observed that the accuracy of the parameters of the multiple linear regression prediction model is low, which may be due to the non-linear relationship between the input parameters and the output parameters. Feng Jinping et al. [61] analyzed the parameters of the Kostiakov three-parameter model by establishing a multivariate linear forecast model. The prediction accuracy is within the allowable range, but the equation can hardly reflect the nonlinear nature of the parameters. Since then, the research results of many scholars have shown that the linear regression prediction model of soil water infiltration parameters is simple in form, convenient in calculation, and widely used. However, most of the prediction accuracy is not high and cannot reflect the true relationship between input parameters and output infiltration parameters.

nonlinear regression prediction model for soil water infiltration parameters
Because linear regression models often do not conform to actual infiltration conditions, relevant scholars have begun to conduct research on nonlinear regression analysis. Saxton [62] and Minasny [63] studied the functional relationship between soil physical and chemical parameters including texture, bulk density, organic matter, etc., and infiltration parameters under different soil matrix potentials. The analysis found that the nonlinear forecast model can make more accurate predictions on the measured values. In 2010, Liao Kaihua et al. [64] substituted the conventional physical and chemical parameters of soil into the nonlinear regression model and found that the nonlinear fitting curve was closer to the measured curve, and the model prediction accuracy was higher. Analysis found that the nonlinear fitting curve is closer to the measured curve, and the model prediction accuracy is higher. Since then, the research results of many scholars have shown that the non-linear regression forecast model of soil water infiltration parameters has high prediction accuracy and can truly reflect the non-linear nature of input parameters and output infiltration parameters.

prediction model of soil water infiltration parameters based on artificial neural network
In 1986, scientists such as Rumelhart and McClelland proposed the concept of BP (back propagation) neural network, which is a multilayer feedforward neural network trained according to the error back propagation algorithm [65]. In 2003, Wang Zhi qiang [66] established a function model of sandy soil conventional physical and chemical parameters and infiltration water holding parameters. In the modeling stage, the nonlinear regression model is better than the BP model. But in the verification stage, the effect of the BP model is better than the prediction effect of the nonlinear model, and the stability is stronger. In 2013, Yuan Lin Hu [67] established the BP model of Philip model parameters.
The prediction effect of each soil water infiltration parameter is better than the regression models. Since then, the research results of many scholars have shown that the artificial neural network prediction model of soil water infiltration parameters has the highest prediction accuracy. But the disadvantage is that the calculation is too complicated, and often does not achieve the global optimal effect, which is not conducive to practical application.

Conclusions and prospects
As an important link in the transformation of the "five waters" and a key factor affecting the growth and development of crops in farmland, the research on soil infiltration has achieved many results. However, due to the particularity and complexity of soil water movement characteristics, there are still many deficiencies in the study of soil water infiltration, which needs to be further improved. Combining the existing research results at home and abroad, the current problems in soil infiltration research and future research directions are mainly reflected in the following aspects.

the mechanism of enhancing soil chemistry effect on infiltration characteristics.
The current research on soil moisture infiltration is mainly about the influence of the basic physical and chemical properties of soil on the infiltration process and infiltration capacity, but there are few studies on the influence mechanism of soil chemistry [44]. Due to the complexity of the distribution and movement of soil water, research on the chemical properties of the earth's soil water (such as the composition and migration of elements) has been slow. Therefore, research on the influence mechanism of soil water sampling methods and soil chemistry in the infiltration process needs to be strengthened [3].

the mechanism of enhancing soil water infiltration on ecological restoration.
With the progress of society and the growth of population, the water ecological environment is further degraded [5]. However, the current research on soil water infiltration is mostly focused on the impact on crop growth and the efficient use of irrigation water, while the research on the impact of infiltration on pollution control and ecological restoration is scarce. Therefore, in the future environment where pollution and ecological problems become more acute, from the perspective of soil water, it is an effective way to strengthen the study of the mechanism of soil water infiltration on ecological restoration and pollution control [3].

strengthen the study of soil water infiltration process under the coupling of multiple factors.
Most of the traditional soil water infiltration studies consider the influence of three factors (such as temperature, water quality, soil bulk density, etc.) on the infiltration process, and most of them are single-point experimental observations. The research on the influence mechanism still needs to be improved. However, soil infiltration in the actual ecological environment is a very complicated process. Any changes in environmental factors and soil factors may affect the dynamic movement of soil water. Future research on the infiltration process should strengthen the connection with the SPAC (Soil-Plant-Atmosphere Continuum) system, and fully consider the mutual feedback of all relevant material information during the infiltration process and the dynamic impact of the environment. This can make the research results closer to the real soil infiltration environment, and can provide a more scientific and reasonable parameter basis for agricultural production practices.

strengthen long-term monitoring of soil water movement in large scale watershed areas
The current human agricultural planting scale is getting larger and larger. However, current soil water infiltration research experiments are mainly local single-point experiments, with small spatial scales and short time spans, which are not representative. Therefore, future research on soil moisture infiltration should be based on 3S technology and big data statistical analysis technology to build a large-scale long-term dynamic monitoring platform for soil moisture infiltration. At the macro level, strengthen long-term research on the process and nature of soil water movement in a wide range of watersheds.

strengthen the prediction model of soil water infiltration.
Due to various limiting factors, it is very difficult to obtain scientific and accurate soil water infiltration parameters through field tests. Therefore, in the future development of more refined and data-based agriculture, the establishment of higher prediction accuracy infiltration parameter prediction models is the future research and development direction. This is of great significance to water and soil conservation and the development of more advanced and efficient irrigation techniques and equipment.


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Effect of Zinc, Iron and their Methods of Application on Growth and Yield of Pearlmillet (Pennisetum glaucum L.)

Pearlmillet occupies fourth position among cereals crops next to rice, wheat and sorghum. Pearlmillet may be an alternative crop that exhibits great advantages in physiological characteristics when compared to other cereals as it is drought resistant, high salinity and high temperature tolerance (Rai et al., 2008). Pearlmillet is the only cereal crop that is capable of producing a reliable yield under marginal environments and simultaneously responds to high management practices. It is high nutritionally better than many cereals as it is a good source of protein (12.6%), Iron (2.8%), zinc and fat (5%) and it is also rich in various vitamins.

Introduction
Pearlmillet occupies fourth position among cereals crops next to rice, wheat and sorghum. Pearlmillet may be an alternative crop that exhibits great advantages in physiological characteristics when compared to other cereals as it is drought resistant, high salinity and high temperature tolerance (Rai et al., 2008). Pearlmillet is the only cereal crop that is capable of producing a reliable yield under marginal environments and simultaneously responds to high management practices. It is high nutritionally better than many cereals as it is a good source of protein (12.6%), Iron (2.8%), zinc and fat (5%) and it is also rich in various vitamins.
The importance of micronutrients has been realized during the past four decades when wide spread of micronutrient deficiencies particularly that of Zinc and iron were observed in most of the soils of the country. Micronutrients have not only cured nutritional disorder in plants but are also known to improve the yield and quality (Jakhar et al., 2006). Zinc being one of the essential micronutrient plays significant role in various enzymatic and physiological activities. It is required as a structural component of large The field experiment was conducted during Kharif 2019 at Central Crop Research Farm, Department of Agronomy, SHUATS, Prayagraj (U.P.) with the objective to study the effect of Zinc, Iron and their methods of application on growth and yield of Pearlmillet (Pennisetum glaucum L.) under Randomised block design comprising of 10 treatments of which soil application of zinc (25 kg/ha) and iron (10 kg/ha) at basal and foliar application of 0.5% of Zinc and iron at 35 and 55 days after sowing which are replicated thrice. The experimental results revealed maximum plant height (156.31 cm), dry weight (49.23g), grain yield (2327.37 kg/ha), stover yield (4584.86 kg/ha) and test weight (7.63 g) was obtained by the application of T 9 (0.5% ZnSO 4 and FeSO 4 foliar spray at 35 and 55 DAS).
number of proteins, such as transcription factors and metallo enzymes (Singh and Kumar, 2009).
Iron is a constituent of several enzymes and some pigments and assists in nitrate and sulphate reduction and energy production within the plant.Iron is also a structural component of porphyrin, cytochromes, ferrchrome. Iron in chloroplasts reflects the presence of cytochromes which is performing various photosynthetic reduction process.
Application of micronutrient fertilizers through soil application is the most efficient and economical method of getting these nutrients into the crops. The amount of nutrients required is much higher compared to foliar application. Foliar application is widely used to apply nutrients for many crops. The deficiency symptoms are usually corrected with in few days (Mortvedt, 2000).

Materials and Methods
The experiment was conducted during Kharif season 2019, at the Crop Research farm, Department of Agronomy, SHUATS, Prayagraj (U.P). The soil of the experimental plot was sandy loam in texture, nearly neutral in soil reaction (7.10), available N (171 kg/ha), available P (15.20 kg/ha), available K (232.50 kg/ha). The treatment consists of soil application of zinc (25 kg/ha) and iron (10 kg/ha) as basal and foliar application of 0.5% ZnSO 4 and FeSO 4 at 35 and 55 days after sowing. There were 10 treatments each replicated thrice. The experiment was laid out in Randomised Block Design. The crop was sown on 23 July 2019 at a spacing of 45cm*15 cm. The recommended dose of 80 kg N, 40 kg P, 40 kg K per ha was applied according to treatment details through urea, DAP, MOP and zinc and iron applied as zinc sulphate and iron sulphate. The half dose of nitrogen and full dose of phosphorous, potassium, zinc and iron were applied as basal. The split dose of nitrogen applied at 50 days after sowing and foliar application of zinc and iron were done at 35 and 55 days after sowing a per treatment details. Five random plants were selected from each plot to record observations on plant growth attributes. Similarly, five random plant samples were collected from each plot at the time of harvest for recording observations on plant yield attributes. Experimental data collected was subjected to statistical analysis by adopting Fishers method of Analysis of Variance (ANOVA) as outlined by Gomez and Gomez (2010). Critical Difference (CD) value were calculated whenever the 'F' test was found significant at 5% level.

Results and Discussion
The growth parameters like plant height and dry weight was significantly affected by the different methods of application of zinc and iron at different stages were represented in Table 1.

Plant height (cm)
The Pearlmillet crop fertilized with 0.5 % ZnSO 4 and FeSO 4 foliar spray at 35 and 55 DAS significantly resulted maximum plant height (156.31cm) which was statistically at par with 0.5% ZnSO 4 and FeSO 4 foliar spray at 35 DAS (154.71cm) at 80 days after sowing. The increase in plant height may be due to zinc plays a key role in metabolic activity and physiology reaction and acts as catalyzing enzymes, transformation of carbohydrates, chlorophyll content and protein synthesis Srinivasan (1992). The iron also had its role in starch formation, protein synthesis and synthesis of chlorophyll in plants. The increase in iron availability to plant might have stimulated the metabolic and enzymatic activities there by increasing the crop growth Trivedi et al., (2011).

Dry weight (g) per plant
The analysed data presented shown significant variation among all other treatments. At 80 DAS significantly maximum plant dry weight (49.23g) were recorded in 0.5% foliar spray of ZnSO 4 and FeSO 4 at 35 and 55 DAS. Which was statistically with Soil application of 25 kg/ha ZnSO 4 +10 kg/ha FeSO 4 (44.82g) and 0.5% ZnSO 4 and FeSO 4 foliar spray at 35 DAS (46.84g).
The increase in plant dry weight might be due to Zinc is the essential component of cellular growth, oxygen synthesis, differentiation and metabolism which results in vigorous growth of plants and extensive root system leading to increased growth of plants reported by Tamil et al., (2019) and Meena et al (2010). The foliar application of Zinc and Iron was found to be more effective due to its higher uptake efficiency compared to soil application as they helps in increased photosynthetic efficiency by delaying leaf senescence reported by Kuldeep et al., (2018).

Yield and Yield Attributes
The yield attributes like test weight, grain yield, stover yield was significantly affected by the different methods of application of zinc and iron at different stages were represented in Table 1.

Test weight (g)
The test weight showed significantly maximum test weight (7.63g) were recorded in 0.5 % ZnSO 4 +FeSO 4 foliar spray at 35 and 55 DAS which was statistically at par with 0.5% ZnSO 4 + FeSO 4 foliar spray at 35 DAS (7.2g). The reason for recording maximum test weight might due to the presence of Zinc in the foliar application stimulates the metabolic processes in seed Qudratullah et al., (2016) and Iron plays role in increasing supply of photosynthates to sink due to higher chlorophyll content and photosynthesis due to foliar sprays at different intervals during growing period of crop. The involvement of the sprayed zinc in enzyme activation, membrane integrity and starch utilization at early which enhanced accumulation of assimilates in the grains Abid et al., (2019).

Grain yield (Kg/ha)
The highest grain yield (2327.37 kg/ha) was recorded significantly superior in 0.5% ZnSO 4 and 0.5% FeSO 4 foliar sprayat 35 and 55 DAS over rest all other treatments. The results might be due to zinc and iron helps in vigorous root development, which promotes growth and development of plant leading to higher photosynthetic efficiency which resulted in better development of higher yield attributes Paramasivan et al., (2011).
The Zinc application might be attributed to its direct role on auxin production, which enables the plant to produce more dry matter and consequently enhanced the partitioning of photosynthates towards newly formed sink. Similar results were observed by Abdul et al., (2018) and Jyothi et al., (2015). Iron is involved in the reductive assimilation pathway of sulphur as ferredoxin is a reductant. Similar results were observed in Yadav and Chippa (2007) and Sareen and Sharma (2010).

Stover yield (Kg/ha)
Stover yield data showing that significant variation among all the treatments. The highest Stover yield was recorded significantly in 0.5% ZnSO 4 and 0.5% FeSO 4 foliar spray at 35 and 55 DAS which was at par with 0.5% ZnSO 4 and 0.5% FeSO 4 at 35 DAS. . Zinc application improves the source and sinks relationship due to translocation of photosynthates which leads to increase of stover yield in Pearl millet Pradeep et al., (2014). Similar results were obtained by Abid et al., (2019) and Sareen and Sharma (2010).
More over quantity of nutrients absorbed due to soil application of Zinc and Iron may not be sufficient to meet the crop demands at seed formation stage. Supplementing the nutrients through foliage at flowering might have the better nutrient balance and there by regaining the photosynthetic efficiency of the plant at post anthesis period results in increased yield Inayat et al., (2015). Foliar application of Zinc and Iron at reproductive stages increased the grain and stover yield might be due to existence of favourable nutritional environment below and above ground Chowdhary (2014).
In conclusion the light of the above study, application of 0.5% ZnSO 4 and FeSO 4 foliar spray at 35 and 55 DAS is more productive.


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Possible causes and the molecular basis of hydrogen cyanogenesis production in cassava

Cassava ( Manihot esculenta ), is a major source of carbohydrates after rice and maize providing a basic diet to over half a billion people. It is an annual crop belonging to the family Euphorbiaceae. It produces edible root tubers which form the staple food for inhabitants in the developing world mainly in the tropical and subtropical countries. It is a very drought tolerant crop which is classified as either bitter or sweet cassava. However, it produces hydrogen cyanide (HCN) which is toxic. This antinutritional component can cause partial paralysis and have been known to kill and wipe out whole families in Africa. It is surprising that farmers seem to prefer the bitter varieties as they are starchier, deter pests and wild animals. There have not been adequate studies to evaluate the causes and molecular basis of the production of Hydrogen cyanide by cassava. This paper highlights the causes of HCN in cassava and ways of minimizing it for food security.

Introduction
Cassava (Manihot esculenta, Crantz), is globally the third largest source of carbohydrates after rice and maize providing a basic diet to over half a billion people. It is an annual rustic crop belonging to the dicotyledonous family Euphorbiaceae (Allem, 2002). It produces edible root tubers which form staple food for inhabitants in developing world mainly in the tropical and subtropical countries (Burns et.al. 2010). It is a very drought and acid soil tolerant crop (Java Cassava, 2007) which although it has thousands of cultivars, it can be classified as either bitter or sweet cassava (Wheatley et al., 1993). The bitterness indicates a physical warning of the presence of a poisonous substance which scientifically has been identified as hydrogen cyanide (HCN) (King and Bradbury, 1995). The cassava cultivars differ very little genetically for they are clonally propagated through cuttings, but surprisingly, studies show however that they have a wide variation in the concentration of HCN ranging from 1 to 2,000 mg/kg (Cardoso et al., 2005, CIAT 2007). The HCN is an antinutritional component which can cause partial paralysis and have been known to kill and wipe out whole families in Africa (Cassava Cyanide Diseases & Neurolathyrism Network (CCDNN), 2011). It is surprising that farmers seem to prefer the bitter varieties as they are starchier, deter pests and wild animals. There have not been many studies to evaluate the causes and molecular basis of the production of Hydrogen cyanide by cassava. This study is designed to attempt to do that. Furthermore, there has been no consensus of the positive correlation between bitterness and HCN level in cassava. For example, Bokanga and Bradbury, 1994, found an almost tasteless cassava variety with more HCN (15 mg of HCN per 100g) compared to a slightly bitter variety with 5mg of HCN per 100g. The problems associated with cassava HCN are not widespread outside Africa (Rosling, 1987), hence the causes of high HCN production should be some unique practices done in Africa. Although, no cassava cultivar, lacks cyanogenic glycosides (Wanda et al., 1998).
The roots and leaves of cassava contain highest amounts of two cyanogenic glucosides (Sinha and Nair, 1968, Jorgensen et al., 2005, Cereda and Mattos, 1996 called linamarin and lotaustralin. The two are broken down by an enzyme called linamarase to produce HCN (Uyoh et al., 2007). However, leaves have a higher cyanogenic glycoside level of 5.0 g linamarin per kg of fresh weight, whereas roots have about twenty times lower. Tribes that traditionally consume cassava have come up with some methods of reducing HCN like soaking, cooking and fermentation, etc. Such tribes have also great ideas of how the HCN is produced by the cassava. This study attempts to reconcile this knowledge with the molecular basis of bitterness in cassava.
Previous studies to explore ways to minimize the cyanide content in cassava and its products had been undertaken but have focused mainly on agronomic factors such as the genotype or cultivar, stress, soil type, fertilization, processing techniques, such as cooking, soaking, fermenting and drying and finally harvest or postharvest practices such as age at harvest, housing of products, storage time and temperature. The above should be combined with other recent advances in plant defense mechanisms and epigenetic studies. This is can be used to decipher the molecular basis of the cause of HCN production in cassava and enhance safety is this important diet crop.

The mechanisms of HCN production
The cyanogenic glycosides are enzymatically hydrolyzed by beta-glucosidase as the cassava root tissues are squeezed during chewing or in the intestine as they are being broken down by gut microorganisms to release HCN which harmful to the consuming predator or human (Poulton, 1988). Studies have shown that HCN producing plants should remain relatively free of damage by general herbivores (Schappert & Shore, 1999;Viette et al., 2000), but can still be attacked by specialists (Glander et al., 1989;Ferreira et al., 1997). In all studies done, it is becoming clear that cyanogenic glycoside and its corresponding cyanogenic enzymes are localized in different cellular compartments or tissues. Therefore, this prevents mixing and cyanogenesis until the tissues are disrupted (White et al., 1998).

Separation and mixing of glucoside and linamarin in cassava
The separation of the substrate and cyanogenic enzymes in some plants is at the subcellular level while in others like sorghum is at the tissue level (Wajant et al., 1994). For example, in rubber trees, the endosperm contains linamarin but the linamarase is located in the apoplast. (Poulton, 1990;Selmar, 1993).
In cassava leaves, linamarin is located in the vacuoles, while the enzyme linamarase is localized to cell walls and laticifers almost 8-fold (Mkpong et al., 1990;Pancoro and Hughes, 1992;Hughes et al., 1994;McMahon et al., 1995;White et al., 1994;White and Sayre, 1995). These results suggest that for HCN to be released there should be a mechanical disruption strong enough to trigger the mixing. Previous studies have shown that linamarin and its βglucosidase, linamarase, are actually present in all cassava organs except seeds (McMahon et al., 1995). An explanation of this substances being located in the cell wall is to offer a physical deterrence against attackers who are trying to gain entry into the cell, but if they enter the cell then the cell triggers another chemical defense against them. This seems to be similar to the two lines of defense of animals' cells against pathogens, the latter being antibodies production. The cassava peel which account for 11% -20 % of the root weight is made up of sclerenchyma and phloem cells; it has a high amount of cyanogenic glycoside and is therefore removed during cassava processing (Sayre et al, 2011) by almost all consumers. The nature and amount of preformed pathogen inhibitors are influenced by the environment, genotype and age of the plant (Price et al., 1987;Davis, 1991).

The possible epigenetic link of HCN release in cassava
Studies by White at al., 1998 suggested that, the molecular basis for the absence of hydroxynitrile lyase, which catalyse the last step to release HCN from roots and stems could be attributed to very low steady-state hydroxynitrile lyase transcript levels (relative to leaves), suggesting that hydroxynitrile lyase expression is regulated at a pretranslational level. However, later studies confirmed that the mechanical disruption could be responsible for its release, for example, it could be found in leaves which are always disturbed compared to stem and leaves in studies in cassava, sorghum and flax (Linum usitatissimum) (Wajant et al., 1994). Later studies could however not fully establish whether linamarin is transported apoplastically between shoots and roots or between root cells (White et al., 1998). Other similar studies further point to the epigenetic expression due to biotic stress, for example, the expression of the rice pir gene is induced during fungal infection. Plants seems to release the enzymes based on some epigenetic memory of the stress using epigenetic processes, like include inherited DNA methylation and histone modifications (Chinnusamy and Zhu 2009), in subsequent cassava generations (Chitwood and Timmermans 2010). Epigenomic control modulates gene expression in response to environmental stimuli through signal transduction and other rapid defenses responses. Cassava has been classified as sweet and bitter cultivars; this demarcation can also be related to the production of defense chemicals (cyanogenesis) by the plants against herbivores and pathogens at the same time. In places with mixed farming the cassava plant might be in close proximity with other plants which attract many microbes, this might make the cassava to produce more defense chemicals than one which is grown in monoculture systems. A cassava in poor soils or harsh environment might also be targeted by pathogens and hence it might produce more toxic defense chemicals. This might explain some cases of cassava poisoning in east Africa region compared to west African region.

The rapid response of cassava to abiotic stresses
The cassava plant opens its stomata only at low evaporation demand and when water use efficiency is highest. The leaves show heloitropic responses making it to obtain maximum light (Berg et al., 1986). The leaves also droop at bright noon light to protect it from excess UV light. (Catalayud et al., 2000;El Sharkawy, 2006). It is no surprise if it has internal mechanisms to protect itself from predators including root pathogens like Phytophthora root rots (PRR; Phytophthora spp. (Edison, 2002).
Cyanogenic glycosides are used by many plants to defend themselves (Francisco and Pinotti, 2000). They also regulate the plant-insect interactions (Zagrobelny et al., 2004). There are at least 2500 species of plants that produce cyanogenic glycosides and a corresponding hydrolytic enzyme called beta-glycosidase. The plantpredator protection mechanism occurs when the two produce a sugar and a cyanohydrin which rapidly decomposed to HCN and an aldehyde or a ketone (Hosel, 1981;Moller and Seigler, 1999). Three, glycosides, cyanohydrins and hydrogen cyanide are known as cyanogens (Moller and Seigler, 1999).

The method of harvesting and hydrogen cyanide production
Cassava is generally manually harvested. The stems are cut off 40 -60 cm above the soil so that the stem portion can be handled when uprooting the tubers. In other cases, harvesting involves digging up the roots (Java Cassava, 2007). The correlation between the harvesting method and amount of HCN has not so far been investigated.

Methodology
This study was done at Mua Hills in Machakos County, Kenya. The lower sides towards Kapiti plains usually has farmers planting cassava but the farmers are harassed by porcupines from the neat by game reserve. The study observed the predatory mechanism of porcupines on cassava and recorded observation in form of photos. The study also focused on literature on cassava on world wide scale. We analyzed and explained some previous results based on recent findings and observations.

Animals studies on Cassava HCN
Our studies with porcupines and cassava farms in Mua Hills in Machakos County, Kenya, showed that when a porcupine dug the roots of cassava and consumed a portion of the root, the porcupine did not return to the same plant the next day. The most likely explanation was that the cassava produced HCN to deter the porcupine from coming to finish its stored food. The porcupine could have the ability to sniff high dangerous concentrations of HCN. This made it to go and dig another plant (Figure 1).

Studies in cassava farms
Our studies showed disturbed cassava farms produced bitter roots, for example when animals passed through a cassava farm the cassava dug from the farm where mostly bitter, when one dug cassava roots immediately after a shower the roots where majorly bitter, also when one hung clothes on cassava plants and latter dug the roots the plants had bitter roots. Surprisingly, when small children struggled to get a cassava root and spent more time trying to get the root out, the roots turned to be bitter. Our Literature studies found out that two families of Makueni District and another of Kathonzweni District, in Kenya were affected after consuming raw and cooked cassava in August and September 2011. A 4-year-old child died in the first family, the family looked extremely poor and the only meal they had was boiled and raw cassava. In the second family, a child aged 5 died in Makueni District Hospital while continuing with management. Both families complained of headaches, abdominal pains and discomfort vomiting, general body weakness and some fever. The Health Officer collected the cooked and uncooked cassava and fresh samples from the same plants where cassava was harvested. The cassava tasted bitter as claimed by the family members. The area had experienced drought for the last 3 years (Cassava Cyanide Diseases & Neurolathyrism Network, 2011).

The time required for a cassava to produce HCN
In removing HCN people usually soak the root tubers for 4 hours but that is not sufficient, only 18-24 hours can reduce HCN by 50%. A dose of 0.5 to 3.5 mg per kg of human body weight is enough to show HCN intoxication symptoms like rapid respiration, low blood pressure, headache and dizziness, intestinal pain, vomiting and diarrhea and can result in death. The mechanical shaking of a cassava plant is easily transferred to the roots as the plant has heavy leaves which are close to the ground where the tubers are formed (Figure 2).

Figure 2
: Cassava tuber exposed due to disturbance by animals and humans. This shows that little disturbance by animals and humans can reduce the probability of cassava plants producing lethal doses of HCN. The soft soil above enabled a porcupine to at least eat a portion of the cassava before the plant started to epigenetically defend itself.

HCN studies on food products
Literature studies showed that samples from Vanuatu had HCN levels of 26 mg/kg to 78 mg/kg but the flour sample from the same had more cyanide content of 57 mg/kg while the cassava chips had 60 mg/kg. When cassava is stored under ambient temperatures, the cyanide levels drops by about 30% after four days (Dolodolotawake and William, 2011). Other studies in Africa show a seasonal variation in cassava HCN levels with higher levels in dry conditions with the cassava becoming bitter. The cyanide content was found to be higher in younger leaves compared to older ones (Hidayat et al., 2002).

Time dependent production of HCN during the harvest period of cassava
The production of Cyanogenesis in cassava can be seen first as a static protection offered by a particular cultivar's constitutive level of cyanogenic glycosides which causes it to have a certain level of bitter taste. Secondly, it can be viewed as a rapid formation of HCN during a mechanical disturbance or a feeding episode by chewing animals or insects on leaves. The first production of bitter glucosides is cultivar, level of growth and other environmental factors, but the second one is a kind of an epigenetic regulation which is rapid as catalyzed by endogenous enzymes to produce HCN. This can help us to understand why some cassava cultivars are mildly bitter but may not be toxic at the level of HCN production depending of the level of disturbance just before harvesting. Some specialized predators like insects have enzymes that transform cyanogenic glycosides into harmless substance in their gut (Fitzgerald et al., 2002) or may hinder the conversion of glucosides in to HCN in their gut, while those who are not adapted to the toxic have to chew it in a way that make it to release the HCN which is lost into the atmosphere before they swallow (Alonso-amelot et al., 2006). Human being depends of the second method to mechanically reduce the HCN before consuming. Therefore, the various processing techniques of cassava significantly reduces the toxicity of HCN because studies have shown that the proportion of HCN, diffused and ingested, will depend on HCN evolution by the plant's tissue, the speed at which the root tuber is eaten. Amazingly, the HCN is also harmful to the plant; therefore it must be produced at a rapid speed at the time of attack or disturbance. Other noncyanogenic aminoacid precursors have been used by plants to deter predators during seed germination and early seedling growth. This phenomenon has been observed in other plants for example in Pteridium arachnoideum (Alonso-Amelot and Oliveros, 2005; Eucalyptus polyanthemos (Goodger et al., 2002) and in the legume Phaseolus lunatus (Ballhorn et al., 2005). The number of cyanogenic glycosides varies in different plant tissues, organs, species and environmental conditions where it grows (Gleadow & Woodrow, 2000). Normally, human beings have acidic stomach environments that deactivate the β-glucosidase enzyme making the production of HCN not possible. How it is that diversification of diet may reduce HCN poisoning? In humans, HCN is detoxified by the enzyme rhodanese, forming thiocyanate, which is excreted in the urine. However, this detoxification used Sulphur donors, which are derived from Sulphur amino acids from the protein rich food consumed.

The epigenetic link of HCN production and food security
The above studies strongly suggest that cassava uses an internal molecular mechanism to protect itself from herbivores and other enemies. It stays alert by preparing a precursor for HCN which through a rapid epigenetic mechanism it triggers an expression of the genes of HCN metabolic pathway when mechanically disturbed. Some cassava plants do not find it necessary to constantly produce the HCN precursor because they are in favourable conditions for a long time. Hence, as we plan to utilize this dry land resource for food security, we should be aware of this by avoiding the HCN from the cassava plant through careful harvesting, processing and habitat selection.