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Comprehensive Genomic Analysis and Expression Profiling of the C2H2 Zinc Finger Protein Family under Abiotic Stresses in Cucumber (Cucumis sativus L.)

Cucumber is one of the most important vegetables in the world. The C2H2 zinc finger protein (C2H2-ZFP) family plays an important role in the growth development and abiotic stress responses of plants. However, there have been no systematic studies on cucumber. In this study, we performed a genome-wide study of C2H2-ZFP genes and analyzed their chromosomal location, gene structure, conservation motif, and transcriptional expression. In total, 101 putative cucumber C2H2-ZFP genes were identified and divided into six groups (I–VI). RNA-seq transcriptome data on different organs revealed temporal and spatial expression specificity of the C2H2-ZFP genes. Expression analysis of sixteen selected C2H2-ZFP genes in response to cold, drought, salt, and abscisic acid (ABA) treatments by real-time quantitative polymerase chain reaction showed that C2H2-ZFP genes may be involved in different signaling pathways. These results provide valuable information for studying the function of cucumber C2H2-ZFP genes in the future.

Introduction
Transcription factors play an important role in plant growth development and abiotic stress responses. More than 60 transcription factor families have been reported in plants [1]. Among them, the zinc finger protein (ZFP) family is widely distributed in the genome of eukaryotes [2,3]. The first zinc finger transcription factor to be found was TFIIIA in Xenopus oocytes [4]. According to the number and position of Cys (cysteine) and His (histidine) residues, zinc finger proteins can be classified into C2H2, C4, C6, C4HC3, C3HC4, C2HC, C3H, and other types [5]. C2H2-ZFP, also known as the TFIIIA type, is currently the most widely distributed and well-studied class of ZFPs in eukaryotic genomes [6].
Recent studies have shown that C2H2-ZFPs play important roles in plant growth development and tolerance to adverse stress. A number of C2H2-ZFPs from rice and Arabidopsis are implicated in trichome initiation, seed germination, floral organogenesis, leaf initiation, and lateral shoot initiation [18][19][20][21][22][23]. C2H2-ZFPs are also involved in the following plant biotic and abiotic stresses: cold, salt, drought, and abscisic acid (ABA) stress in rice and Arabidopsis [24][25][26][27]; cold stress in banana [28]; cold and drought stress in soybean [29]; and biotic stress in potato [11]. These studies demonstrate that C2H2-ZFPs are involved in multiple growth processes and stress responses in plants.
Cucumber is one of the most important vegetables in the world. Due to the diversity of cucumber flower sexual types, it has become an ideal model for studying the sex differentiation mechanism [30,31]. Although C2H2-ZFPs are important, only one cucumber C2H2-ZFP has been reported to date [32]. Fortunately, the cucumber genome has been reported [33], which provides us with an opportunity to investigate C2H2-ZFPs.
In the present study, we identified 101 C2H2-ZFP genes from the cucumber genome and analyzed their chromosomal location, gene structure, conservation motif, and phylogenetic relationship. In addition, we also analyzed C2H2-ZFP gene expression profiles in different organs of cucumber. Some of them were selected as stress-related candidate genes to be tested under different abiotic conditions using real-time quantitative polymerase chain reaction (RT-qPCR). These results should provide valuable information for studying the function of cucumber C2H2-ZFPs in the future.

Chromosomal Location and Phylogenetic Analysis
Information on the distribution of cucumber C2H2-ZFP genes on the chromosomes was obtained using the TBtools software (GitHub, San Francisco, CA, USA). Cucumber and Arabidopsis C2H2-ZFP protein sequences were used for phylogenetic analysis. The MEGAX software was used to construct a phylogenetic tree using the maximum likelihood (ML) method with partial deletion of 1000 bootstraps and a WAG model. The results were formatted for display using Evolview V3 [40,41].

Collinearity Analysis
Gene duplication analysis of Trihelix genes in different species was performed using the Multiple Collinearity Scan software toolkit (MCScanX) (Plant Genome Mapping Laboratory, University of Georgia, Athens, GA, USA) with default parameters [42]. We plotted the collinearity relationship of the C2H2-ZFP genes from selected species using the TBtools software.

Gene Structure and Motif Analysis
The gene structure of cucumber C2H2-ZFP genes was identified via TBtools. The online Multiple Expectation Maximization for Motif Elicitation (MEME) program version 5.0.5 [43] was used to identify conserved motifs of the cucumber C2H2-ZFPs [44].

Expression Profile Analysis
To analyze the expression profiles of cucumber C2H2-ZFP genes in different organs, we retrieved public RNA-seq data (accession number: SRP071224) from the National Center for Biotechnology Information's Short Read Archive database. Analysis of RNA-Seq data from 23 sampled cucumber tissues was performed based on the regular protocol by Yang et al. [45]. The heatmap of cucumber C2H2-ZFP gene expression profiles was generated using the TBtools software.

Expression Analysis of Abiotic-Stress-Responsive C2H2-ZFP Genes in Cucumber
Cucumbers from the inbred line 9930 were used as plant materials. The experiment was implemented at Shanghai Jiao Tong University (Shanghai, China). Plants were grown in a growth room at day/night temperatures of 24/18 • C with a 16 h photoperiod. When the cucumber seedlings were at the five-true leaf stage, we conducted the following treatments: 100 mM NaCl, 100 g/L Polyethylene glycol (PEG) 6000, 100 µM ABA, and 4 • C. The NaCl and PEG6000 were added into a nutrient solution and hormones were sprayed onto the leaves. For cold treatment, cucumber seedlings were moved into an illuminated incubator. Leaves from three different plants were harvested at 0, 1, 3, 6, 12, and 24 h after treatment. Each experiment was repeated at least three times. All samples were frozen with liquid N 2 and stored at −80 • C for RNA isolation.
Total RNA was isolated from leaves using an OminiPlant RNA Kit (CWBIO, Beijing, China). Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using a HiFiScript cDNA Synthesis Kit (CWBIO, Beijing, China). Quantitative real-time PCR was carried out using the UltraSYBR Mixture (CWBIO, Beijing, China) PCR reactions were prepared and performed according to the manufacturers' protocol. The β-actin gene (GenBank: AB010922) of cucumber was used as an internal control. Geneious software was used to design primers according to the cDNA sequences (Table S1). PCR-amplified product lengths were 100-150 bp. The analysis of relative microRNA (mRNA) expression data was performed using the 2 −∆∆Ct method [46]. Each expression profile was independently verified in three replicate experiments performed under identical conditions.

Identification of C2H2-ZFP Genes in Cucumber
A two BLAST method was used to identify cucumber C2H2-ZFPs using sequences of Arabidopsis C2H2-ZFPs as a query sequence. We found 105 candidate genes, and all putative genes were subsequently verified in the SMART and Pfam databases to confirm the existence of the C2H2-ZFP domains. In total, 101 C2H2-ZFP genes were detected and verified.
The basic information for the 101 C2H2-ZFP genes of cucumber is provided in Table S2, including coding sequence (CDS), amino acid sequence, isoelectric point (pI), and molecular weight (MW). Csa5G477610 was the smallest protein with 103 amino acids, whereas Csa6G487810 was the largest protein with 1464 amino acids. The protein MWs varied from 11.31 kDa (Csa1G657510) to 164.89 kDa (Csa6G487810), and the pIs ranged from 4.97 (Csa6G509590) to 9.96 (Csa6G312040).

Chromosomal Distributions and Phylogenetic Analysis
The 101 C2H2-ZFP genes are distributed across seven cucumber chromosomes, ranging from 9 to 24 genes per chromosome ( Figure 1). Chromosome (Chr) 3, the longest chromosome, also contained the highest number of C2H2-ZFP genes. Chr2 was longer than Chr7 but contained the least number of C2H2-ZFP genes. To investigate the evolutionary relationship of C2H2-ZFP genes, a phylogenetic tree was constructed using full-length C2H2-ZFP protein sequences of cucumber and Arabidopsis ( Figure 2). The C2H2-ZFPs from both species were divided into six groups named I to VI. Groups I and II were the biggest groups and included 27 and 26 members, respectively. The smallest group (Group VI) had seven members from cucumber. To investigate the evolutionary relationship of C2H2-ZFP genes, a phylogenetic tree was constructed using full-length C2H2-ZFP protein sequences of cucumber and Arabidopsis ( Figure 2). The C2H2-ZFPs from both species were divided into six groups named I to VI. Groups I and II were the biggest groups and included 27 and 26 members, respectively. The smallest group (Group VI) had seven members from cucumber.

Figure 2.
Phylogenetic analysis of C2H2-ZFPs in Arabidopsis and cucumber. The phylogenetic tree was based on C2H2-ZFP sequences from cucumber (101 proteins, marked with a black dot) and Arabidopsis (208 proteins). Each of the six C2H2-ZFP groups is indicated in a specific color.

Collinearity Analysis of the Relationship among Cucumber, Melon(Cucumis melo), and Arabidopsis Members
We performed a comparative analysis to identify the collinear C2H2-ZFP genes among melon, cucumber, and Arabidopsis ( Figure 3). Only 15 genes from cucumber shared collinear relationships with Arabidopsis. C2H2-ZFP genes on cucumber chromosome 1 showed no collinearity with Arabidopsis. However, the collinear relationships of C2H2-ZFP genes between cucumber and melon were quite rich. In total, 79 C2H2-ZFP genes in cucumber were collinear in melon (Table S3). These results suggest that C2H2-ZFP genes in cucumber and melon have strong relationships.

Collinearity Analysis of the Relationship among Cucumber, Melon(Cucumis melo), and Arabidopsis Members
We performed a comparative analysis to identify the collinear C2H2-ZFP genes among melon, cucumber, and Arabidopsis ( Figure 3). Only 15 genes from cucumber shared collinear relationships with Arabidopsis. C2H2-ZFP genes on cucumber chromosome 1 showed no collinearity with Arabidopsis. However, the collinear relationships of C2H2-ZFP genes between cucumber and melon were quite rich. In total, 79 C2H2-ZFP genes in cucumber were collinear in melon (Table S3). These results suggest that C2H2-ZFP genes in cucumber and melon have strong relationships.

C2H2-ZFP Gene Structures and Conserved Motifs
To better understand the phylogenetic relationships between the structure and function among cucumber C2H2-ZFP genes, the conserved motifs and exon/intron structure were analyzed ( Figure  4). In the phylogenetic tree, all 101 C2H2-ZFP genes were divided into six groups, namely I, II, III, IV, V, and VI, containing 17, 14, 15, 26, 17, and 22 members, respectively. We identified ten conserved motifs among the 101 C2H2-ZFPs (Table S4). Domain 1 includes a plant-specific conserved sequence ('QALGGH'), which was present in 85 proteins. In addition, the most closely related members had a common motif composition. For example, motifs 1, 2, 3, 4, and 5 were observed in most group I members. Motifs 6 and 8 were specifically detected in group III. The great majority of group IV members only contained motif 1.
Analysis of the structure of C2H2-ZFP genes also produced a similar result. C2H2-ZFP genes in the same group shared similar numbers of exons/introns. For example, most members in groups IV and V had one exon, while all members in group I had more than two exons.

C2H2-ZFP Gene Structures and Conserved Motifs
To better understand the phylogenetic relationships between the structure and function among cucumber C2H2-ZFP genes, the conserved motifs and exon/intron structure were analyzed ( Figure 4). In the phylogenetic tree, all 101 C2H2-ZFP genes were divided into six groups, namely I, II, III, IV, V, and VI, containing 17, 14, 15, 26, 17, and 22 members, respectively. We identified ten conserved motifs among the 101 C2H2-ZFPs (Table S4). Domain 1 includes a plant-specific conserved sequence ('QALGGH'), which was present in 85 proteins. In addition, the most closely related members had a common motif composition. For example, motifs 1, 2, 3, 4, and 5 were observed in most group I members. Motifs 6 and 8 were specifically detected in group III. The great majority of group IV members only contained motif 1.
Analysis of the structure of C2H2-ZFP genes also produced a similar result. C2H2-ZFP genes in the same group shared similar numbers of exons/introns. For example, most members in groups IV and V had one exon, while all members in group I had more than two exons.

Expression Profiles of Cucumber C2H2-ZFP Genes in Different Tissues
To analyze the tissue specificity in cucumber, a heatmap of 89 cucumber C2H2-ZFP gene expression profiles in 23 cucumber tissues was generated ( Figure 5, Table S5).
The results showed that the C2H2-ZFP genes presented diverse expression profiles among different tissues, implying distinct roles for various developmental stages. The gene expression levels including Csa2G354820, Csa7G428820, Csa3G697940, Csa1G613510, Csa7G428830, Csa4G642460, Csa6G452080, Csa6G152950, Csa1G657150, and Csa4G290810 were consistently high in all organs. In contrast, Csa3G902400, Csa3G141870, Csa4G290830, Csa5G372190, Csa7G024090, Csa5G365160, Csa7G071620, Csa4G095040, and Csa5G606610 were barely expressed in all organs. In addition, several genes were observed in specific tissues as follows: Csa1G132120 showed specific expression in roots and hypocotyl; and Csa3G199020 had the strongest expression in flowers. These genes may be involved in the development of the corresponding phenotype.

Expression Profiles of Cucumber C2H2-ZFP Genes in Different Tissues
To analyze the tissue specificity in cucumber, a heatmap of 89 cucumber C2H2-ZFP gene expression profiles in 23 cucumber tissues was generated ( Figure 5, Table S5).

Expression Analysis of C2H2-ZFP Genes under Abiotic Stress
To demonstrate if cucumber C2H2-ZFP genes are involved in stress tolerance or response to hormones, 16 cucumber homologous genes of Arabidopsis abiotic-stress-responsive genes were selected.
The expression patterns of 16 C2H2-ZFP genes in leaf tissues were analyzed under low temperature (4 • C), drought (PEG), NaCl, and ABA treatments. As shown in Figure 6, all sixteen C2H2-ZFP genes were induced by at least one treatment.
Under cold stress treatment, ten genes were induced. The transcription of four genes (Csa1G043020, Csa2G033890, Csa3G743410, and Csa4G006510) was inhibited, whereas the transcript level of the other six genes was increased. The six upregulated genes were induced significantly at 24 h after cold treatment. Eight genes were mediated by PEG treatment as follows: seven genes were upregulated, and one gene (Csa3G743410) was downregulated. The same eight genes were upregulated after NaCl treatment. The transcript of Csa3G829160 was strongly upregulated (>20-fold) at 6 h after NaCl treatment. Only five genes were mediated by ABA treatment as follows: four genes were upregulated, and one gene (Csa3G117400) was downregulated.
Among the 16 C2H2-ZFP genes, only one gene (Csa3G697940) responded to all four treatments, and two genes (Csa2G033890 and Csa2G382580) responded to three treatments. The remaining thirteen genes were mediated by one or two treatments. The expression profile indicated that C2H2-ZFP genes may be involved in cucumber responses to abiotic stress, especially to low temperature, high temperature, and NaCl treatment. Figure 6. Expression analysis of 16 selected C2H2-ZFP genes in leaf tissues under cold, drought, salt, and abscisic acid (ABA) stresses using qRT-PCR. The relative microRNA (mRNA) abundance of 16 selected C2HC2-ZFP genes was normalized with respect to the reference β-actin gene (GenBank AB010922). The x-axis represents time points after stress treatments. The y-axis is the scale of the relative transcript abundance level. Error bars represent the standard deviations from three biological replicates. Asterisks indicate stress treatment groups that showed a significant difference in transcript abundance compared to the control group (* P < 0.05 and ** P < 0.01). Figure 6. Expression analysis of 16 selected C2H2-ZFP genes in leaf tissues under cold, drought, salt, and abscisic acid (ABA) stresses using qRT-PCR. The relative microRNA (mRNA) abundance of 16 selected C2HC2-ZFP genes was normalized with respect to the reference β-actin gene (GenBank AB010922). The x-axis represents time points after stress treatments. The y-axis is the scale of the relative transcript abundance level. Error bars represent the standard deviations from three biological replicates. Asterisks indicate stress treatment groups that showed a significant difference in transcript abundance compared to the control group (* P < 0.05 and ** P < 0.01).

Discussion
C2H2-ZFPs mainly participate in growth development and abiotic stress responses of plants as thoroughly studied in rice [16], durum wheat [17], and poplar plants [15,47]. However few studies have investigated C2H2-ZFP genes in cucumber. In this study, we identified a total of 101 cucumber C2H2-ZFP genes. The C2H2-ZFP gene family has 176 members in Arabidopsis. The genome of cucumber (genome size of 367 Mb) is larger than the genome of Arabidopsis (genome size of 125 Mb) [33], but the number of ZFPs identified in this study is less in cucumber than in Arabidopsis. Whole-genome duplication (WGD) is common in angiosperm plants and produces a tremendous source of raw material for gene genesis. Previous research has revealed three WGD events in Arabidopsis. A paleohexaploidy (γ) event occurred in Arabidopsis after the divergence of monocotyledons and dicotyledons. Subsequently, two WGDs (α and β) occurred in Arabidopsis. The recent WGD events have played an important role in the rapid expansion of gene families [48]. However, cucumber lacks recent WGD events [33,49], resulting in the low number of cucumber C2H2-ZFP genes.
The collinearity analysis revealed that there are only 16 pairs of homologous genes in cucumber and Arabidopsis but that more orthologous gene pairs are present in cucumber and melon (Figure 3), which is consistent with the phylogenetic relationship among cucumber, melon, and Arabidopsis. According to the number of melon and cucumber homologous genes, we found that cucumber chromosomes 1, 2, 3, 5, and 6 were collinear to melon chromosomes 2/12, 3/5, 4/6, 9/10, and 8/11, respectively, indicating that these cucumber chromosomes each resulted from a fusion of two ancestral chromosomes after speciation [33].
To analyze the evolutionary relationship of cucumber C2H2-ZFPs, a phylogenetic tree was constructed. In Figure 2, we found cucumber's Tu (Csa5G577350) gene to be closely associated with Arabidopsis ATZFP6 (AT1G67030). Tu is implicated in cucumber fruit tuberculate development [32]. AtZFP6 plays a key role in regulating trichome development [50]. This result revealed that C2H2 genes may have conserved functions in the same group.
Generally, C2H2-ZFPs with a similar number of exons and motif composition were classified in the same groups ( Figure 4). The domains and motifs of transcription factors are often related to protein interaction, transcriptional activity, and DNA binding [51]. The motif analysis showed that most of the C2H2-ZFPs of cucumber contained the Q-type motif 1, 'QALGGH', plant-specific sequence. In our study, approximately 84% (85 out of 101) of the cucumber C2H2-ZFPs belonged to Q-type C2H2-ZFPs, which is a large number compared to that of Arabidopsis (34%, 64 out of 176) [6] and rice (52%, 99 out of 189) [16], but similar to that of durum wheat (79%, 96 out of 122) [17]. Studies have shown that residues of the Q-type motif play an important role in binding activity [52]. Motifs 6 and 8 were specifically detected in group III, indicating that they play an important role in this subfamily. These results suggest that although some motifs of the C2H2-ZFPs are highly conserved, the new evolutionary motifs may perform new functions in some plants and that the function of these motifs requires further verification.
C2H2-ZFP genes play a role in specific tissues at certain stages of plant growth development. Transcriptome data from different tissues and periods were used to analyze the expression profiles of cucumber C2H2-ZFPs. Csa6G303740 had the strongest expression in roots. ZAT11, a homologous gene of Csa6G303740, was highly expressed in roots and particularly in root tips [53]. Csa7G428260 transcript signals were detected only in flesh. MaC2H2-1/2 (two C2H2-ZFPs) is involved in the regulation of fruit ripening in banana via transcriptional repression of ethylene biosynthetic genes [28]. These results may reveal the similar biological roles of these genes in different species. Csa3G199020 showed specific expression in flowers. AtZFP1, an Arabidopsis ortholog of Csa3G199020, is highly expressed in the shoot apex and plays an important role in shoot development [20]. These results suggest that their functions may vary in different plant species.
Arabidopsis Transgenic ATHB7:: GUS plants showed no or very low GUS activity in all tissues under optimal water conditions. However, the GUS activity was obviously induced in plants subjected to a limited water supply [54]. ATHB12, a paralogous gene of ATHB7, has a similar expression pattern and function [55]. Some genes were expressed at high levels in each tissue throughout the cucumber developmental stages, which suggests that these genes may be involved in the entire life cycle of cucumber. Although some genes were rarely expressed in all tissues, it did not indicate that they had no function in cucumber. It is possible that these genes may be expressed under specific environmental conditions.
Many studies have shown that C2H2-ZFP genes respond to abiotic stress and their expression changes after different time points after stress treatment [15,47,56]. In our study, 16 genes were induced by at least one type of stress. Ten genes were induced by cold treatment, indicating that cucumber C2H2-ZFP genes may be more sensitive to low temperatures. Csa3G697940 was induced by all treatments. Correspondingly, Arabidopsis AtSAP5, a homologous Csa3G697940 gene, also responded to environmental challenges, including salt, osmotic, cold, and ABA stress [8]. Previous studies have shown that osmotic stress response is highly related to ABA response [56]. In our study, five genes were induced by ABA stress, three of which also responded to NaCl stress. These results demonstrate that zinc finger proteins may be involved in multiple environmental stresses and are worthy of further investigation in the future.

Conclusions
In this study, 101 cucumber C2H2-ZFP genes were identified and divided into six groups (I-VI). Chromosomal location revealed that the 101 cucumber C2H2-ZFP genes were distributed in all cucumber chromosomes. C2H2-ZFPs with a similar number of exons and motif composition were classified in the same groups. RNA-seq transcriptome data of different organs revealed temporal and spatial expression specificity of the C2H2-ZFP genes. Expression analysis of sixteen selected C2H2-ZFP genes in response to cold, drought, salt, and ABA treatments by RT-qPCR showed that C2H2-ZFP genes may be involved in different signaling pathways. These results provide valuable information for studying the function of cucumber C2H2-ZFP genes in the future.

Conflicts of Interest:
The authors declare no conflicts of interest. The sponsors had no role in the design, execution, interpretation, or writing of the study.


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

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Effect of Sulphur and Phosphorus on the Growth and Yield of Black Gram (Vigna mungo L.)

A field experiment was carried out during kharif season of 2019 at Research Farm, of School of Agriculture, ITM University, Gwalior. The experimental work was conducted in randomized block design with three replications. The treatment comprises of three levels of phosphorus (0, 20 and 40 kg P2O5 ha -1 ) and three levels of Sulphur. Growth parameters viz. plant height, number of branches, number of leaves plant -1 and dry matter yield plant -1 appreciably improved with the application of phosphorus up to 40 kg P2O5 ha -1 . More values of these parameters were appreciably higher with the application of 15 and 30 kg S ha -1 over the control. The root length, total and effective number of nodules plant -1 and fresh and dry weight of root nodules were significantly higher with the application of 40 kg P2O5 ha -1 while sulphur had no significant impact on these parameters. All the yield attributes, biological yield and grain yield appreciably increased with increasing rates of phosphorus as well as sulphur application. Economics revealed that, highest net return (Rs.75870.4) and benefit cost ratio (3.42) was observed with I2 while, highest net return (Rs.49153 ha -1 ) and B:C ratio (3.32) were observed with application of 40 kg P2O5 and 30 Kg S ha -1 .

INTRODUCTION
India is the world's largest producer as well as consumer of black gram. It produces about 1.5 to 1.9 mt of urad annually from about 3.5 m ha of area, with an average productivity of 500 kg ha -1 . Black gram output accounts for about 10% of India's total pulse production. Black gram production in the country remained stable more than a decade through the 2000s at around 12 to 14 lakh tons. But a sudden jump in output was noted in 2010-11 to 17.5 lakh tonnes primarily on account of rise in production from Madhya Pradesh, Rajasthan and Tamil Nadu. As per the latest available estimates, UP and Maharashtra occupy the first two positions, contributing over 32%. Individually, Madhya Pradesh and Andhra Pradesh contribute 14% each to total production in the country.
Agricultural feasibility and economic viability of black gram (Vigna mungo L.) as sole as well as intercrop in different crop sequence have been established due to its unique property of maintaining and restoring soil fertility through biological nitrogen fixation as well as conserving and improving physical properties of soil by virtue of its deep root system and leaf fall.
Phosphorus is essential constituent of every living cell. It is concerned with structural compounds, nucleic acids for reproductive purpose, and the conservation and transfer of energy in the metabolic reactions taking place. Phosphorus stimulates the symbiotic nitrogen fixation because in presence of phosphorus bacterial cell becomes mobile which is pre-requisite for migration of bacterial cell to root hair for nodulation (Charel 2006). It also plays an important role in the process of photosynthesis, energy conservation and transportation, cell division and meristematic growth in living tissues, grain quality and most of physico-biochemical activities.
Sulphur is another essential nutrient which is usually required by leguminous crops in amounts comparable to Phosphorus. Sulphur is known to help in chlorophyll formation, stimulating growth, seed formation and Nitrogen fixation by enhancing nodule formation.
Unlike Nitrogen, Sulphurdeficiency symptoms appear first on the younger leaves, and will persist even after Nitrogen application. Plants deficient in Sulphur are small and spindly with short and slender stalks, growth is retarded, maturity in cereals is delayed, nodulation in legumes may be poor and Nitrogen fixation reduced. Sulphur application also has marked effect on soil properties and is used as soil amendment to improve the availability of other nutrients in soil as gypsum and pyrite (Nad & Goswami, 2010). Keeping the above facts in view the present study was conducted in Kharif season of 2018 at Research Farm, School of Agriculture, ITM University to study the effect of P and S, on growth, yield and economics of black gram.

MATERIALS AND METHODS
A field experiment was conducted on sandy loam soil at Research Farm, of School of Agriculture, ITM University, Gwalior, during kharif season of 2018. The experiment comprising of three levels of phosphorus (0,20 and 40 kg P 2 O 5 ha -1 ) and three levels of Sulphur (0,15 and 30 kg S ha -1 ) in Black gram. Thus, in all 9 treatment combinations were compared in a 'Randomized Block design' having with three replications. The soil was deficient in available nitrogen (183.50 kg ha -1 ) and medium in available phosphorus (14.4 kg P 2 O 5 ha -1 ) and available potash (243.00 kg K 2 O ha -1 ).
Black gram variety Awasthiwas sown with seed rate of20 kg ha -1 in furrows at the depth of approximately 3-4 cm by hand and covered with soil immediately. The recommended quantities of fertilizer (20 kg N and 20 kg K 2 O ha -1 ) and levels of P and S as per treatment were applied through Urea, Single Super Phosphate, Muriate of potash (MOP) and Sulphur (Cossavet), respectively. Entire required quantity of nitrogen, phosphorus, potash and sulphur were applied at the time of sowing, as basal application. The irrigation was applied as per requirement of the crop on different growth stages and other management practices were adopted as per recommendations.

RESULTS AND DISCUSSION
The plant growth attributes i.e. plant height, number of branches and number of leaves plant -1 appreciably improved with the application of phosphorus up to 40 kg P 2 O 5 ha -1 . The application of phosphorus significantly increased dry matter accumulation in plant at all the stages of crop growth and differences were well marked up to 40 kg P 2 O 5 ha -1 . The magnitude of increase in dry matter yield plant -1 with 20 and 40 kg P 2 O 5 ha -1 was to the tune of 13.66 and 21.16 per cent, respectively over the control. The favourable effect of phosphorus on growth might be due to the fact that phosphorus is chief constituent of the lipoids and nucleo proteins, an abundance of phosphorus in the meristemmatic region might have helped in cell division and multiplication, it is also concerned the carbohydrate transformation, respiration and nitrogen fixation and hence boosted plant growth. The response of phosphorus application in terms of dry matter accumulation in plant up to 40 kg P 2 O 5 ha -1 have also been reported by Niraj and Ved Prakash (2014). Application of 15 and 30 kg S ha -1 resulted in appreciably higher dry matter plant -1 then the control at all the stages of crop growth. At harvest, the increase in dry matter accumulation plant -1 with the application of 15 and 30 kg S ha -1 was 13.23 and 21.05 per cent, respectively over the control. Dry matter accumulation is the resultant of better plant growth. In this study, plant height, number of branches plant -1 and number of leaves plant -1 also exhibit similar trend to that of recorded in dry matter accumulation in plant. Niraj and Ved Prakash (2014) also reported significant increment plant height, number of branches plant -1 and dry matter accumulation in plant of black with the application of 30 or 40 kg S ha -1 over the control.
The plant with 20 and 40 kg P 2 O 5 ha -1 took about 49 and 47 days for 75 per cent flowering and about 85 and 82 days for maturity, respectively. However, application of sulphur extended about one day to 75 per cent flowering and maturity. The root length, total and effective number of nodules plant -1 and fresh and dry weight of root nodules were significantly increased with every increase in the rate of phosphorus application up to 40 kg P 2 O 5 ha -1 .However, Application of sulphur had no significant impact on these parameters. The beneficial effect of phosphorus on root development and nodulation were also reported by Kumar and Singh (2008).
Biological yield ha -1 appreciably increased with increasing rates of phosphorus application up to 40 kg P 2 O 5 ha -1 . The magnitude of increase in biological yield with 20 and 40 kg P 2 O 5 ha -1 was to the tune of 86.47 and 134.64 per cent, respectively over the control. Which may be ascribed to the favourable effect of phosphorus application in case of plant height, number of branches and leaves plant -1 . These results are in closed conformity with those of Kumawat, et al. (2013) and Niraj and Ved Prakash (2014).
Grain yield consequently increases with every increase in the rate of phosphorus application up to 40 kg. P 2 O 5 ha -1 . The magnitude of increase in grain yield with 20 and 40 kg P 2 O 5 ha -1 was to the tune of 104.58 and 176.5 per cent, respectively over the control. The application of phosphorus increases the symbiotic nitrogen fixation power and in turn, increased number of pods plant -1 , Length of pod, number of grain pod -1 and 1000 grain weight and ultimately grain yield.
Biological yield of black gram appreciably increased with increasing rates of sulphur application. The increase in biological yield with 15 and 30 kg S ha -1 was to the tune of 59.80 and 101.46 per cent, respectively over the control. Grain yield significantly increased with every increase in the rate of sulphur application up to 30 kg S ha -1 . The increase in grain yield with 154 and 30 kg S ha -1 was to the tune of 69.95 and 110.34 per cent, respectively over the control. Black gram crop responded well to the application of sulphur up to 30 or 40 kg S ha -1 have also been reported by Niraj and Ved Prakash (2014).
The interaction effect of phosphorus and sulphur levels was found to be significant on grain yield of black gram. Black gram crop fertilized with phosphorus at 40 kg P 2 O 5 ha -1 along with 30 kg S ha -1 was the best combination for higher grain production in Gwalior conditions. Harvest index significantly increased up to 40 kg P 2 O 5 ha -1 , because it is the function of seed yield and biological yield and these two yields were increased up to 40 kg P 2 O 5 ha -1 . 30 kg S ha -1 appreciably improved harvest index over other levels of sulphur application and control. Nita chand et al. (2002) and Kumawat et al. (2013) have also reported likewise. In this experiment the highest net profit of and B: C ratio was recorded with the application of 40 kg P 2 O 5 and 30 Kg S ha -1 followed by 20 kg P 2 O 5 and 30 Kg S ha -1 . Similar findings were observed by Niraj and Prakash (2014).   CONCLUSION On the basis of the results summarized above, it is concluded that Plant and root growth and yield and yield attributes can be obtained appreciably higher with the application of 40 kg P 2 O 5 ha -1 and 30 Kg S ha -1 .On the basis of


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IN VITRO ELIMINATION OF DAHLIA MOSAIC VIRUS BY USING MERISTEM CULTURE, ELECTROTHERAPY AND CHEMOTHERAPY

This study was conducted to investigate the effect of different in vitro techniques on elimination of dahlia mosaic virus. Meristems of Dahlia mosaic virus (DMV) infected dahlias, with 0.2-0.3 mm in length, were cultured on Murashige and Skoog (MS) medium. Based on DAS-ELISA, 100% of the survived plantlets by meristem culture were virus-free. DMV infected stem segments with two axillary buds were treated at three different levels of electric currents (15, 25 and 35 mA) and two time courses (10 and 20 min) in an electrophoresis tank and cultured on MS medium. The treatment of 35 mA for 20 min was the best for viral elimination from diseased dahlias by 85% followed by 25 mA for 20 min by 70%. Acyclovir and salicylic acid were the antivirals were used to eliminate DMV from the infected dahlias. For chemotherapy, nodes were cultured on MS media supplemented with antivirals at 0, 10, 20, 30, 40 and 50 mg/l for 20 and 30 days. The highest concentration of acyclovir (50 mg/l) for 30 days were the best treatments as its highest effectiveness on elimination ability (90.67%) but gave only 49% for plantlet survival. Whereas 40 mg/l acyclovir for 30 days gave elimination ability (80%) and plantlet survival (89.67%). The therapy containing salicylic acid at 40 mg/l for 30 days was the best as its highest effectiveness on DMV elimination ability (100%). But the concentration of salicylic acid at 30 mg/l for 30 days was the best treatment (chemotherapy efficiency= 49.21) as well as DMV elimination ability (75.33%) and plant survival (65.33%). The in vitro developed dahlia plantlets were tested for DMV-freeness using DAS-ELISA. The using therapies (electrotherapy and chemotherapy) can play a good role in virus elimination because they need the minimum period of time to regenerate fully developed healthy plants as long as 2-3 months.

العراقية الزراعية العلوم مجلة
. DMV virion is icosohedral particle, 48-50 nm in diameter, consists of double stranded DNA (dsDNA) genome with a size about 7 killobase pairs (42). It is well established that the viral diseases in different crops lead quantitative and qualitative losses of the flowers a plant produced (30,35). Virus control is very difficult because virus replication inhibitors have shown to be having toxic effects on the host plant cell. This is the main reason for using of micro-propagation techniques to eliminate virus (9,40). Tip meristem culture is the most commonly used method to obtain plants free of virus (36). Morel and Martin (22) first demonstrated the elimination of viruses from dahlia using meristem culture. Since the use of meristem culture to obtain virus-free ornamental plants has been widely used by numerous groups of researchers (38). Electrotherapy has been applied by using electric currents on infected plant tissues for virus elimination from any part of the infected plants especially the vegetative propagules. The chemicals used in chemotherapy are antivirals incorporated in to culture media with a known capability to stop virus replication and their natural and synthetic varieties are tested for their potential to eliminate DNA and RNA plant viruses (35). The requirements of a useful antiviral chemical is its abilities to inhibit multiplication and spread of the virus; be selective enough not to harm the host plant; have broad-spectrum activities against a number of viral diseases; move systemically in the host; and have not harmful effects on the environment. Sharma et al. (37) studied the effect of acyclovir in vitro that was one of the best chemicals that inhibit virus in the infected plants. Exogenous salicylic acid application induced antioxidant systems to reduce oxidative stresses caused by pathogen infection (41,45) especially viruses (14). Tian et al. (43) found that salicylic acid inhibits the replication of plant viruses. This study was conducted to evaluate the effect of meristem tip, different levels of electric currents and varying concentration levels of antivirals (acyclovir and salicylic acid) in the elimination of Dahlia mosaic virus (DMV) from infected dahlias.

MATERIALS AND METHODS
In spring of 2016 and 2017, young leaf samples of symptomatic dahlia plants (mosaic, systemic vein clearing, necrotic spots, stunting and malformation) collected from various gardens and nurseries of Duhok governorate/ Iraq. The collected leaves were used for virus isolation. The developed plants from cultured dahlia seeds with four fully expanded leaves were inoculated mechanically with virus isolated.

Diagnosis of DMV:
The presence of DMV was detected in infected dahlia leaves using double antibody sandwich (DAS)-ELISA. The DMV antiserum kit was requested from SEDIAG Company, France. The diagnosis of the virus performed according to the manual instructions of kit. DMV elimination Meristem culture: Dahlia shoots that contain 3-5 axillary buds were collected from the greenhouse grown dahlias infected with DMV. The excised shoots were kept under running tap water before sterilization. The binocular microscope was surface sterilized by 70% ethanol. Forceps and scalpels were sterilized on flame burner. The shoot disinfestations were done by using 1% HgCl 2 and 2 drops of Tween-20 and shaking for 5 min, in a laminar airflow cabinet. The treated shoots were washed with sterilized distilled water three times, 5 minutes for each washing. The leaves protecting the meristem were removed and the meristem was excised with 0.2-0.3 mm length under the microscope. The excised meristem contained meristem tip with one or two leaf primordia (Fig. 1). The explants were inoculated in glass jars containing MS medium supplemented with 0.5 mg/l IAA and 0.25 mg/l GA 3 . The developed meristems, after 4 weeks of inoculation, were subcultured into a new media with the same composition. The plantlets when reached to an average length of 5 mm with few leaves were subcultured into multiplication medium supplemented with 0.0, 0.5, 1.0, 1.5 and 2.0 mg/l BA and 0.25 mg/l GA 3 . The plantlets with 3-4 cm height were transferred into a full and half strength media containing different concentration of IBA (0.0, 0.5, 1.0, 1.5 and 2.0 mg/l) to induce rooting (12,27). Well developed plantlets with root systems were taken out from the controlled environment. The plantlets were washed thoroughly under running tap water to remove the medium. Then, the plantlets were transplanted into a sterilized peat-moss in addition of benlite (2g/l) and kept at room temperature for 10 days to contact normal temperature. After hardening-off period, the developed plants transplanted into a tightly controlled atmosphere of the greenhouse (28). The most vigorous stems of DMV infected dahlias were selected and used for electrotherapy. Stem segments with two axillary buds were treated at three different levels of electric currents (15, 25 and 35 mA) and two time courses (10 and 20 min) in an electrophoresis tank (20). Electric currents were supplied by an electrophoresis power supply. The treated axillary buds were removed from stem segments and cultured on MS medium supplemented with BA (2 mg/l) and GA 3 (0.25 mg/l), after sterilizing with 0.1% mercuric chloride for 5 min. Effects of in vitro electrotherapy treatments on virus elimination and regeneration of plantlets were studied using ten replicates. Symptomatology test was used for viral presence in the developed explants. Efficiency of the therapy was estimated according to the following formula: efficiency of therapy = virus elimination percent × percent of plantlet regeneration (32). Chemotherapy: Stem cuttings with one or two leaf nodes cut from DMV infected plants and cultured on MS medium containing two antivirals including acyclovir and salicylic acid (23,33). Acyclovir and salicylic acid were added at 0, 10, 20, 30, 40 and 50 mg/l. The chemicals were applied into MS medium just after autoclaving. Shoots were cultivated on these types of media for 20 and 30 days and transferred into the medium containing plant growth regulators without antivirals. Well developed explants tested for viral presence using symptomatology. Percentage of survived cuttings counted for each concentration after 20 days of treatment (6,47). Efficiency of the therapy was estimated according to the formula mentioned in electrotherapy. The in vitro grown explants were incubated at 25± 2ºC under 16/8 hrs (light/dark) photoperiod provided by fluorescent tubes at 1000 lux light intensity in growth chamber. Treatments were consisted of ten glass jars with three replicates and the factorial experiment within Complete Randomized Design (CRD) was used. All data were statistically analyzed with computer using SAS program (34). The difference s between various treatment means were tested with Duncan Multiple Range test at 5% level.

RESULTS AND DISCUSSION Diagnosis of DMV Using DAS-ELISA
Positive reactions were observed in diagnosis of DMV in infected dahlia leaves and tubers by using DAS-ELISA that was characterized by yellow color as a response of the specific antigen (DMV) to its specific antibody. The colorless columns show the negative reaction that indicated the absence of the virus particles as well as viral infection in the leaves and tubers of the symptomless plants.  Table 1 shows in vitro growth patterns of D. pinnata taken after four weeks of culture. The shoots number and the shoots height were positively correlated with each other where the value of highest mean was recorded in MS medium containing 2.0 mg/l BA and 0.25 mg/l GA 3 (shoots number: 3.14, shoots height: 5.58 cm). The lowest shoots number and the shoots height were recorded in control treatment (1.28 and 2.5 cm). The required time for developing the meristem explants to become rooted plantlets was as long as seven months. The rooted plantlets were successfully acclimatized in sterilized peat-moss with a survival rate of about 95%. The developed plantlets were tested for DMVfreeness using DAS-ELISA. Moreover, the percentage of the virus freeness in regenerated plantlets was 100%. Several techniques were evaluated for the ability to eliminate viruses from infected plants. Effects of meristem culture and electrotherapy were studied comparatively by Pazhohandeh et al. (32) indicating that the efficiency of each technique is dependent upon the cultivar-virus system and the intensity of the virus eradication treatment. After 10-20 days of meristems incubation, they were checked for their response to in vitro culture by gradual increasing in size and color change. The observation showed that the color of the grown meristems changed to green due to chlorophyll development. Disappearance of green color indicated the absence of chlorophyll development. Addition of growth regulators to MS medium plays an important role in shoot growth (9). Mehmood et al. (18) found that Plantlets developed in M.S media supplemented with 0.25 mg/l of GA 3 produce maximum plant height, more number of nodes, reduced no. of days to root initiation and took less number of days to transferable height of the plant. Different combinations of GA 3 and IAA within MS medium influenced in vitro shoot regeneration of meristem tip culture. This combination showed the best result for tip meristem development and shoot length improvement (6). Ibrahim and Daraj (2015) found that 2.0 mg/l BA gave the highest significant response to shoot formation. The inclusion of auxins, like IBA, in culture medium has a positive effect on in vitro rooting of the shoots (39). Depending on the results, DMV could not introduce meristem with 0.2-0.3 mm height. Similar results have been noticed by Sedivá et al. (38) who eradicated DMV from infected plants by meristem-tip culture. Hull (11) reported that 0.2-0.3 mm meristem-tips with two leaf primordia were used for virus elimination. He reported that if the taken tissues include slightly expanded leaf primordia, the survival and growth of meristems would be greatly improved but at the same time increased the chance of virus entrance. Meristem culture provides an economical and reproducible method for producing virus-free plants. As meristem-tips are free from pathogens, virus eradication and regeneration of virus-free plants are possible through this technique. The efficiency of virus elimination will be different based on the virus and its host genotype (19). The principle of this technique depends on that many viruses are unable to infect the meristematic cells of the growing points of the plants. It remains still unclear that why the meristem has no virus, so there are several suggestions that may responsible of high protection against viruses. In meristematic cells, high metabolic activity and chromosome replication during mitosis may inhibit virus multiplication through interference with the metabolism of viral nucleic acid, so the virus may lose its ability to replicate (25). Meristematic tissues have the highest auxin concentration than other plant tissues. It has been suggested that auxin may inhibit the multiplication of the virus (10). Vascular tissue is a supporting element for rapid dissemination of viruses. Because of the absence of differentiated cells, the viruses that infect plant systemically have no ability to enter the meristematic tissues. Gene silencing, the process that is very active in meristematic tissues, is a mechanism that acts to defense the plant against viruses. This phenomenon may act as a restrict mechanism to inhibit the virus multiplication (1,11). In general, the small size of the meristem increases the chance of virus elimination while the larger sizes increase the survival chances (15,21). Meristems smaller than 0.2 mm have no ability to survive and those larger than 0.7 mm produce plants carrying virus (1). Tip-meristems larger than 0.5 mm were used in combination with some therapies such as electrotherapy (17). Electrotherapy Plants were regenerated successfully from all electrotherapy treatments. The treatment of 35 mA for 20 min was generally the most effective among the treatments in elimination of DMV at 85% and then 25 mA for 20 min at 70%. The treatments of 15 mA for 10 and 20 min had no effect on virus elimination (Tab. 3). Regenerated control plantlets without electrotherapy treatment were positive for DMV (Tab. 3). Depending on the formula (Efficiency of therapy= virus elimination percent× plantlet regeneration percent/ 100), the efficiency of electrotherapy was estimated (32). The treatments of 35 mA for 20 and 10 min were the most efficient (55.25% and 52.50%), respectively, among the treatments in regenerating the in vitro cultured dahlias and also in eradicating of DMV. The required time for developing the treated explants to become rooted plantlets was as long as 2-3 months.  Earlier attempts to eliminate some plant viruses using different electric current of 7 (46) and 15 mA (5) resulted in decreasing the level of virus titer in regenerated plantlets. Despite of the level and duration of electrotherapy, and plant genotype the efficiency of electrotherapy may be affected by structural differences between plant viruses as well as DMV has an isometric body. In addition to the limitations of conventional methods in virus elimination, their efficiency is also relatively low when compared with electrotherapy (32). Regenerated control explants without electrotherapy were tested positive for virus indicating that diminishing virus titer was due to electrotherapy and not to the in vitro regeneration. The electrotherapy technique described here was successful in eliminating dahlia mosaic virus. For production of virus-free plantlets, in addition to virus elimination, the efficiency of plant regeneration is also important because the adverse effects of electrotherapy may hamper the growth of dahlia plants (32). The regeneration rate of the treated plantlets was generally negatively affected by increasing the intensity of electrotherapy power, in such a way the highest regeneration was observed in treatment of 15 mA for 10 min, followed by 25 mA for 10 mA and 35 mA for 10 min. Treatment of 35 mA for 20 min led to the lowest regeneration percentage of 65% while showing the highest virus elimination rate of 85% (Tab. 3). Similar levels of regeneration have also been reported in earlier studies, including regeneration rates of 40 to 100% (5,32).
There were no noticeable morphological differences among the regenerated plantlets (31). Electrotherapy is a simple and faster protocol of virus eradication, compared to conventional virus elimination techniques, without the need to use of any especial or expensive equipment (32). Based on our results, this method can be used as an effective technique for virus eliminating. Conditions of electrotherapy may differ depending on host-virus system, so it is important to determine the optimal electrotherapy treatment conditions for a desired host-virus system.Further studies are necessary to investigate the possible use of this protocol in other plant species especially vegetatively propagated ornamental plants.

Chemotherapy
The concentration of each antiviral influenced the number of the survived plants. There was a significant difference between the treatments in the number of the regenerated plants.
Depending on the results showed in Tab.4 and 5, the proportion of the obtained virus-free plants was increased by increasing the concentration of both antivirals. The highest concentration of acyclovir (50 mg/l) for 20 and 30 days were the best treatments as its highest effectiveness on elimination ability (90.67%). On the other hand, depending on chemotherapy efficiency, 40 mg/l of acyclovir for 30 days (the longest incubation period) was the best treatment (71.73%) as its highest effectiveness on elimination ability (80%) and plantlet survival (89.67%). The result was close to that of Dewanti et al. (5) who found that acyclovir produces 100% in vitro virus free plantlets.     Effect of chemotherapy on DMV elimination from infected dahlia explants was influenced by the used antiviral compounds and the levels of concentration. Toxicity to plants and antiviral activity was more pronounced in higher concentrations of 50 and 30mg/l of both acyclovir and salicylic acid respectively as evidenced by discoloration of leaves. This high toxicity resulted into high mortality of plants and then low survival rate of cultured explants. Increasing in acyclovir concentrations typically increases the effectiveness of virus elimination, but at higher concentrations especially at 50mg/l slowed plant growth and phytotoxicity was evident. The required concentrations of many antivirals, that is necessary to inhibit virus multiplication, are very close to that point induce toxicity for host plants (26) thus at 40mg/l and 30mg/l survival rates decreased slowly but the number of the produced healthy plants was higher for both acyclovir and salicylic acid. In general, antivirals at effective low concentrations hinder virus replication. This concentration however needs to be below as well as acyclovir is not highly toxic to the plant at 40mg/l but its phytotoxicity increased more and more at 50mg/l. Although salicylic acid had the lowest survival rates at 40mg/l, it resulted higher proportion of healthy plants while at 50mg/l it induces high toxicity resulted into full mortality of in vitro cultured explants. Salicylic acid is a phenolic compound synthesized in plant in response to viral infection which is essential for local and systemic resistance (13,44). It plays a vital role in signaling plant resistance to the infection while there is little information about its mechanism (43). Biosynthesis of salicylic acid is activated on recognition of viral effectors by R gene products. Activation of interaction induces several responses to inhibit virus multiplication at the site of infection (3,7). It is also responsible for activation of systemic acquired resistance in distal tissues that reduces the effects of secondary attacks (2). The present study demonstrated that meristem culture, electrotherapy and chemotherapy are the techniques that can be used for in vitro elimination of DMV. Depending on the results and based on the efficiency of the therapies, chemotherapy using acyclovir (40mg/l for 30 days) is the best treatment can be used to produce in vitro DMV-free plants (efficiency of therapy=72%). Then, electrotherapy at 35mA for 20 min gave good results (efficiency of therapy=61.75%) for this purpose. The used therapies can play a good role in virus elimination because they need the minimum period of time to regenerate fully developed healthy plants as long as 2-3 months.


s2fieldsofstudy:
Agricultural And Food Sciences
Economics

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Sustainable Utilization of Indigenous Goats in Southern Africa

Goats have a key role in ensuring food security and economic livelihood to smallholder farmers in rural areas. Women play a vital role in goat rearing, promoting economic autonomy within households. Indigenous goats dominate and are of high significance due to their adaptive traits that are relevant for climate change and low maintenance. However, lack of emphasis on farmer-centered technology development and proper breed characterization remains a hitch to sustainable utilization and breed development of indigenous goats. This can be over come through proper linkage between market and production, workable regional and national agricultural policies, community breeding programs, collaborative research work within the region, and consistent government support.

Introduction
The Southern African region is a host to approximately 38 million goats [1]. About 70% of the goats are kept under traditional management systems where the farm structure comprises of about twenty (±20) goats. Goat productivity in this system is relatively low due to minimal inputs, poor infrastructure, undefined marketing channels and multiple breeding objectives [2,3]. Indigenous goatsare, however, the most popular breed due to their ability to cope with a range of climatic conditions including disease challenges, inadequate feed resources and low management [4]. These genetic resources are more crucial to livelihoods as climate change progresses and environmental changes arise. They provide nutrition and financial stability to poor resource households and play a vital role in the African culture.
Due to their adaptability, indigenous goats are found in a wide range of production environments in Southern Africa. Although goats as a strategic resource have contributed to sustainable utilization of the environment, they have been blamed of causing environmental degradation in other areas [5]. If managed well, due to their smaller size (low maintenance) and their ability to browse in poor and dry areas where other ruminants cannot, goats can contribute significantly to sustainable rangeland utilization. While most of the environmentally harmful effects of goat grazing arise from improper management practices particularly at very high grazing pressures, if managed adequately, goat grazing can be a useful tool for environmental conservation [5]. For example, it has been found that mixed grazing of indigenous cattle and goats at a ratio of 2:1 had a complementary advantage for improved utilization of pasture resources and animal performance compared to when either of the species was kept [6]. The Southern African indigenous goat population is comprised of a mixture of large (e.g., Nguni, Tswana, Matebele) and smaller East African (e.g., Landim, Mashona, Malawian) indigenous goats ( Figure 1). These goats are generally characterized by small body size, slow growth rate, low milk yield and low carcass weight. In order to improve productivity of these goats, cross-breeding with exotic breeds was initiated around the 1980s [7]. Unfortunately, this did not yield any positive results to the farmer's livelihood as it was not coordinatedproperly. There was a mismatch of breeding goals, a failure to capture tangible values and improve the existing management of low input production systems [2]. The most concerning factor included continuation of haphazard crossbreeding and breed replacement practiced by the farmers, which has a negative impact on the existing population of indigenous goats as genetic resources.
Sustainable utilization of indigenous goats is the key for efficient conservation, breed improvement, management, and marketing. This entails comprehensive information on breed identity and their unique eccentrics, as well as their environments. Such information can be attained through characterization studies. In southern Africa, characterization studies have been conducted on production systems [3,8,9], phenotypic characterization [10][11][12][13] and genetic characterization [13][14][15][16]. Initiatives have been made to conserve indigenous goats as purebred flocks in different countries of the region. Programs, structures and modalities to implement conservation with sustainable utilization are, however, still lacking, and this remains a challenge to food security. The aim of this article is to give perspectives on the alternative approaches towards conservation, development and sustainable utilization of indigenous goats in the Southern African region.
Diversity 2020, 12, x FOR PEER REVIEW 2 of 9 had a complementary advantage for improved utilization of pasture resources and animal performance compared to when either of the species was kept [6]. The Southern African indigenous goat population is comprised of a mixture of large (e.g., Nguni, Tswana, Matebele) and smaller East African (e.g., Landim, Mashona, Malawian) indigenous goats ( Figure 1). These goats are generally characterized by small body size, slow growth rate, low milk yield and low carcass weight. In order to improve productivity of these goats, cross-breeding with exotic breeds was initiated around the 1980s [7]. Unfortunately, this did not yield any positive results to the farmer's livelihood as it was not coordinatedproperly. There was a mismatch of breeding goals, a failure to capture tangible values and improve the existing management of low input production systems [2]. The most concerning factor included continuation of haphazard crossbreeding and breed replacement practiced by the farmers, which has a negative impact on the existing population of indigenous goats as genetic resources.
Sustainable utilization of indigenous goats is the key for efficient conservation, breed improvement, management, and marketing. This entails comprehensive information on breed identity and their unique eccentrics, as well as their environments. Such information can be attained through characterization studies. In southern Africa, characterization studies have been conducted on production systems [3,8,9], phenotypic characterization [10][11][12][13] and genetic characterization [13][14][15][16]. Initiatives have been made to conserve indigenous goats as purebred flocks in different countries of the region. Programs, structures and modalities to implement conservation with sustainable utilization are, however, still lacking, and this remains a challenge to food security. The aim of this article is to give perspectives on the alternative approaches towards conservation, development and sustainable utilization of indigenous goats in the Southern African region.

Characterization of Indigenous Goats
Phenotypic characterization is the primary phase for documentation of qualitative and quantitative traits of the indigenous goats [17]. Goats in the region are locally named according to different ethnic names and geographical habitats but show similar phenotypes. They are mostly multi-colored, Diversity 2020, 12, 20 3 of 9 bearded, horned, with medium to broad lopped ears, short hair and can be small to medium size [18]. Most variations observed in phenotypic characterization studies were due to environmental factors, resulting in different ecotypes. These variations have caused some discrepancies with regard to breed and ecotype identification. Although further studies are needed in this regard, genetic characterization is also needed to guide the final decisions on breed improvement and sustainable utilization programs.
Molecular technologies such as mitochondrial DNA, microsatellite markers and single nucleotide polymorphism (SNPs) are important for genetic characterizations [19]. Table 1 shows studies on genetic characterization of indigenous goats in different countries in Southern Africa. These studies have provided information on genetic diversity, genetic structure and genetic relationship within and between populations, which is relevant for breed monitoring and development and for the general improvement of goats. The scarcity of knowledge, however, still remains with genetic linkage of Southern African indigenous goats and adaptive traits that can be exploited for sustainable utilization to improve productivity and contribute to food security.

Sustainable Utilization
Derivation of significant benefits from goats as a strategic genetic resource in Southern Africa is still to be experienced. This is despite the existence of the Global Plan of Action [22] and national plans for management and utilization of genetic resources. While these plans are commendable, their implementation at the rural community level remains a challenge due to a variety of reasons. Therefore, a paradigm shift is required especially with regard to the way we view indigenous goats. Currently, governments, Non-Governmental Organizations (NGOs), markets and development agencies continue to promote exotic goat breeds and crossbreds at the expense of indigenous ones, despite the previous failures. A change of attitude by researchers, policy makers and extension workers towards traditional goat keepers and their breeds is very important. In Southern Africa, the majority (90%) of farmers are categorized as smallholders and own 75 percent of farm animals comprised mainly of indigenous breeds [23]. Consequently, for any production improvement strategies, the need for goat owners to be involved in decision-making and ownership of such initiatives is important. No matter how much effort is put into financial and technological support, the eventual survival of improvement programs depends on whether the farmers understand and agree with the objectives of the projects [24]. With that in mind, farmers' knowledge, innovations and practices must be integrated into research and extension. Community based programs have been found to be integral to the success of sustainable management and utilization of animal genetic resources (AnGR) [25].

Community Based Breeding Program
While indigenous goats have a competitive edge under prevailing climatic and environmental conditions, they are still generally viewed as less productive than exotic breeds. Therefore, practical selection programs are needed to improve productivity and add more value to indigenous goats. Community based breeding program (CBBP) is perceived as a strategy that can support breeding and conservation efforts in rural communities where flock sizes are small, and implementation of recording schemes are almost impossible. Setting up a CBBP usually follows these important steps: Definition of breeding objectives, development of selection criteria, genetic evaluations, selection of animals, design of appropriate mating systems and strategies for dissemination of superior genetics [26], and establishing a monitoring and evaluation framework [27]. Through capacity building and in order to integrate community knowledge in breed identification, farmers are involved in identifying relevant breeding objectives and selection criteria for their animals. Apart from increased production, breeding objectives should be aligned with market requirements in order to improve competitiveness of indigenous goat products as well as to challenges due to climate change by selecting for improved health, functional and other fitness traits. However, in other communities, preferences such as color, behavior and mothering ability can be considered as important [28].
CBBPs rely on the principle of establishing a communally owned open nucleus breeding system from which top breeding males are selected and disseminated to breeding flocks within the community. These males undergo rotational exchange while new ones are continuously selected to replace old ones. The concept has so far, been successfully implemented in sheep and goats in Ethiopia, and out scaled to goats in Uganda and Malawi through projects from the United States Agency for International Development and the United States Department of Agriculture. Other practices include castration of undesirable males, pedigree recording and progeny testing [25,29] and genomic characterization. Genetic progress and economic evaluation of CBBP has been undertaken in Ethiopia [30] and is underway in Uganda and Malawi through various postgraduate studies. Preliminary results show phenotypic increase in growth rates of offspring born from selected bucks. It has been concluded on one site that goats from CBBP sites have higher carcass yields than from non-CBBP sites [31].
Genetic improvement programs can only be implemented where accurate performance and pedigree recording is possible, which has proven unsuccessful in most indigenous goat production systems due to a lack of structures for record-keeping. Therefore, simple and easy to understand data collection tools should be formulated for indigenous goat keepers. Local management of recording schemes should be encouraged through the promotion of farmer groups and cooperative projects [24]. It is advisable to back up the localized recording scheme with an effective extension service with a view of finally mainstreaming the smallholder breeding activities into existing national livestock recording and performance testing schemes.
Since most smallholder farmers keep small flocks, formation of clusters or groups would enable them to acquire improved economies of scale. As a block, they can easily lobby government, undertake group funding or solicit private sector funding for the much needed infrastructure that includes breeding and reproductive technology centers, roads, milk collection facilities etc. Furthermore, they can collectively negotiate better prices from input suppliers, transport providers and at the market. An example is that of a well-detailed CBBP for West African goat in Liberia. The objectives of this CBBP entailed increasing productivity and income of resource-constrained farmers-by providing access to improved animals that respond well to feeding and management-while facilitating and targeting specific market opportunities [32].
Finally, for sustainability and success of these communities based programs, specialized communication strategies should be established, coupled with program monitoring and evaluation. This entails dialogue and feedback as well as review, implementation and revision of the developed programs.

Production Related Issues
Goat keepers in rural communities are faced with challenges at different phases of the goat production value chain. One such constraint is unavailability and high cost of input supplies such as medicine, vaccines and feed to mitigate effects of diseases, internal and external parasites, and seasonal fluctuations in feed quality and quantity [3]. In some cases, due to erratic rainfall and climate change, communities have encountered shortage of drinking water and feed for their animals. Possible interventions to these shortcomings include government incentives through subsidies on veterinary and feed supplies to the resource constrained farmers. Development of a one stop supply center at a strategic location within a community cluster would bring resources closer to farmers. Farmers belonging to a cluster could benefit from resource-sharing such as use of communal boreholes funded through government, donor agencies or cooperatives. The continued existence and success of these clusters relies on the ability of a cluster to source out their funds and not over-rely on donations which is more sustainable, in the long run. Furthermore, crop residue and other locally available feed materials could be used together with commercial feed ingredients to develop more palatable and nutrient balanced mixed rations. This could alleviate malnutrition and mortality in goat production.
Interventions to production constraints should integrate indigenous knowledge from the communities and scientific evaluation from research and academic institutions, with extension services provided by different sectors such as government and NGOs. Inclusion of existing knowledge in indigenous goat production provides an opportunity to better improve community knowledge and encourage participation. Rural communities continue to rely on several indigenous knowledge practices such as breeding practices [23], anthelmintic properties of some fodder crops that can be used to control internal parasites [25] and the ability to recognize changes in their climate and local environment using natural indicators [33]. However, for relevance, broad adoption and competitiveness, indigenous knowledge needs to be enhanced and well packaged for marketing and dissemination purposes.
Information and knowledge has become an important factor in timely decision making for effective livestock production. With advancements in technology, many rural communities have gained access to communication networks especially through mobile telephones and call centers. Therefore, efficient and fast dissemination of information between research, extension, and farmers can be made possible by exploiting such telecommunication facilities. This can be in the form of a mobile phone application (app) on routine goat health and management, or basic telemedicine where a veterinarian may discuss a case with a farmer over the telephone.

Marketing of Indigenous Goats and their Products
Indigenous goats are usually sold live on produce markets, mostly by middlemen who aggregate from farmers in rural areas. Formal channels exist in some countries, however, sales based on dressed weight attract lower gross margins unless sold at premium prices. Indigenous goats have unique characteristics which can be exploited and translated into niche products. These products range from colored hides, extra-fine fiber to meat or milk with special taste [34]. Niche products can be appealing and are generally more expensive than standard products hence they can motivate farmers through increased economic profitability. Therefore, niche products seem to present prospects to conserve indigenous goat breeds in a favorable economic context. Furthermore, due to their unique features, indigenous goats and their products can also play an important role in agrotourism, whereby local and international tourists visit communities where they are kept for viewing, photography and exploration. Traditional processing techniques (those involved in producing handicrafts or garments with distinctive designs) and strong local ties (breeds that are found only in certain localities and are raised by certain ethnic groups) could form the basis of marketing indigenous breeds and their products [34] including for tourism purposes. Markets should be well profiled and production objectives must be geared towards that. This should be consistent and sustainable as it will help in commercializing the small ruminant sector and in changing farmers' mind-set to be market oriented. Moreover, CBBP centers offer market convenience to sell surplus breeding stock to neighboring communities, and non-selected goats to middlemen and butcher men who subsequently take them to a commodity market.

Gender Aspects and Marginalized Groups in Goat Production
Women play a key role in goat value chains by providing much of the needed labor-especially more in goat rearing than other livestock. However, women face greater constraints than men, including economical/financial, social and institutional barriers, which can affect their participation in livestock value chains and the benefits that can be gained from them [35]. These challenges are mainly a consequence of their poor access to land, little involvement in decision-making, credit, markets, assets and technical information and to their stereotyped role within households and communities, particularly in rural settings [36,37]. However, through participatory approaches women can be represented better in community-based AnGR conservation and sustainable utilization programs. Furthermore, there is a need to reduce continued male dominance by developing gender sensitive policy initiatives to ensure that women and men have equal access to land, research and extension services, credit and other facilities [38]. There are marginalized ethnic groups who also play a pivotal role in keeping indigenous goat resources and have ensured their continued existence. However, these groups also suffer the same fate as women. Empowerment and increased involvement from women and marginalized groups will result in input from diverse experiences, household security, cultural backgrounds, strategies and knowledge that can enrich the community based programs.

Regional Policy and Legal Framework
Despite individual country efforts in conservation and management of indigenous goats, more could still be achieved at the regional level. This is advanced by the fact that some goat breeds are common across several countries or at least have common ancestry. However, due to the absence of regional data integration, the genetic relationship of these goat breeds remains largely unknown. In any case, exploration of genetic resources across countries compared to within countries presents an opportunity to access a large pool of genetic diversity with potential for an enormous selection response. Availability and use of AnGR across the countries and beyond, harmonization of common policy, and creation of a user-friendly and accessible regional database haven't really been achieved as previously suggested [39]. Hence, a legal framework to accelerate and enforce implementation of these noble policy aspects is needed. This will also guide important issues such as exchange of genetic resources and samples between countries especially with the advent of shared facilities like regional gene banks and genetic analysis laboratories.

Future Research and Development
Some of the research that could be carried out in future to enhance conservation, utilization and management of indigenous goats include: (1) Determination of risk status of indigenous goat breeds to inform whether a conservation program is necessary or not. Demographic data collection should be carried across the region to obtain information such as genetic distinctiveness and adaptive traits of the goat breeds. (2) The establishment of effective breeding population sizes since they are not known for most indigenous goat breeds. These can be determined by carrying out breed surveys or on the basis of experience or previous census/surveys or by analyzing data stored by countries in the Domestic Animal Diversity Information System (DAD-IS). (3) More data collection in extensive farming systems so that these can be genotyped, analyzed and documented. Genetic diversity analyses and selective signals/sweeps can be identified and association studies can be conducted for various traits including adaptation and disease resistance traits. (4) Meta-analyses of genomic data which has already been collected on different goat breeds within the region [13,15,16]. (5) Establishment, out-scaling and evaluation of community based breeding and development programs for indigenous goats with the aim of improving and conserving the local breeds.

Conclusions
Indigenous goats are important genetic resources kept under resource-poor farming systems with potential for increased productivity to enhance food security and the nation's welfare. Compared to cattle, these genetic resources have become even more important under changing climates.
Sustainable utilization and conservation of these genetic resources should be linked to the market. This will assist in supporting and exploiting niche products efficiently. In each country, there should be regulatory policies that will protect and regulate sustainable utilization of indigenous goats and developmental policies that will provide rules and regulations to facilitate marketing process of indigenous goat products.
The use of genomic tools has the potential to be explored in low-input farming systems by identifying SNPs associated with different traits e.g., disease resistance and adaptation, and these can be used to develop 'customized' chips for the low-input farming systems.
Farmer participation, informed policies and ring-fenced budget allocation to national indigenous genetic resources will promote and ensure sustainable utilization of indigenous goats in the region.
Author Contributions: All authors contributed in writing the review paper. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.


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Impact of various irrigation practices on nitrate movement in soil profile and wheat productivity

Nitrate is important for plant nutrition and is considered as an index of soil fertility. Its leaching to underground water not only poses severe threat to environment but also to human health. Amount of water supplemented to crops have significant role in yield and nitrate distribution in soil. An experiment was carried out to evaluate the effect of different irrigation rates on nitrate movement, soil properties and wheat yield. Experiment was laid out in randomized complete block design with three replicates. Three irrigation levels applied were I1 (0.5 ET0 deficit irrigation), I2 (1.0 ET0; according to farmer’s practice) and I3 (1.5 ET0; over irrigation). Wheat (Sehar-2006) was used as test crop. Irrigation levels had significant effects on most of growth and yield parameters of wheat. Maximum number of leaves per plant, 1000-grain weight and grain yield were observed in second irrigation level. Irrigation rates had significant effect on infiltration rate and soil saturated hydraulic conductivity, and a decrease in value of these parameters was observed in I3 that was mainly due to higher moisture contents. Bulk density and porosity of the soil remained unchanged under various treatment applied.

Introduction
Nitrogen is considered as an essential part of agriculture and has been reported to boost the grain yield of many cereal crops (Ehdaic et al. 2010). Nitrogen can be taken up with water by crops. In modern agriculture, it is fully understood that water and nitrogen have an interactive relationship and irrigation significantly affects nitrogen status in the soil and low efficiency of fertilization (Peoples et al. 1995). Under current agricultural practices, there appears to be a shortage of both water and fertilizer. Meanwhile increasing population and restricted irrigation facilities for agricultural lands always demands a priority for the production of excessive food.
Enhanced N fertilization not only reduced nitrogen use efficacy but also increase the loss of N in the form of NO 3 leaching ultimately polluting the ground water in intensive farming regions (Hu et al. 2005). There are many sources of nitrate to pollute ground water, but agriculture is the most important source of ground water-pollution mostly caused by unnecessary use of N fertilizers; especially in intensive cropping systems like wheat, maize and potato (Ladha et al. 2005). The nitrogenous fertilizers are nitrified into NO 3 , and due to a negative charge (anion) and excessive water application this element could leach down gradually resulting in contaminated groundwater (Spalding and Exner 1993). Nitrate leaching is a multifaceted process and is affected by many factors like N fertilizer content, cropping system, soil type, tillage, irrigation methods and irrigation intensity. Two most important factors are fertilizer and irrigation rates; these both affect nitrate movement in direct way as increasing rates give elevated nitrate leaching. Drinking water is contaminated with nitrate, and this is a growing issue worldwide. Nitrate pollution in ground water was studied in many developed countries and reported as a very severe problem . The presence of high concentrations of nitrate in rivers and oceans cause a negative effect on marine ecosystems, with the production of anoxic zones and eutrophication of aquatic environments (Diaz 2001).
Wheat is a shallow-rooted crop, which can extract nitrate below root zone under greater irrigation and rainfall. From the applied nitrogen to the soil, only 50% is absorbed by plants and the remaining goes to environment through different pathways, i.e., leaching, volatilization and denitrification (Craswell and Godwin 1984). N loss from agricultural land through leaching can reduce crop yields and enhance negative environmental impacts such as eutrophication (Brady and Weil 2008).
Nitrate leaching can be improved by precision agriculture and application of fertilizer according to plant requirement; it was concluded by Marcelino et al. (2011) after determining the role of precision agriculture in reduction in nitrate leaching. Excessive use of N from optimum level causes greater NO 3 − losses, and it is related to applying N and its uptake in grain (Lord and Mitchell 1998). Split application and optimum amount (180 kg N ha −1 ) of fertilizer lowered the leaching loses (Liang et al. 2011). This study was conducted with following objectives; (1) to quantify NO 3 − movement in soil profile at different irrigation levels, (2) to assess the irrigation level where NO 3 − leaching was minimum without compromising the yield, (3) to check the effect of irrigation on soil physical properties and to evaluate the impact of irrigation on crop yield.

Materials and methods
A field study was conducted at research farm of Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad, Pakistan to evaluate the impact of various irrigation practices on nitrate movement in soil, soil properties and wheat yield. The soil of the experimental area was poor in NP contents and was calcareous in nature having pH 7.8. Prior to experimentation, soil samples were collected for nitrate concentration, organic carbon contents. The soil physical properties (soil bulk density, saturated hydraulic conductivity, infiltration rate) were also measured by using standard methods (Table 1). Soil was prepared for seed bed by applying two planking's followed by two ploughings. The experiment was laid out in randomized complete block design (RCBD) having three replications. There were three irrigation rates I 1 = 0.5 ET 0 , I 2 = 1.0 ET 0 and I 3 = 1.5 ET 0 . Wheat variety (Sehar 2006) was sown as test crop. The seed rate 125 kg ha −1 was used. Recommended doses of N/P/K 110, 90 and 60 kg ha −1 were applied, respectively. At maturity plant height was measured. Plant samples were also collected from each plot according to standard procedure.
Soil bulk density was determined by core sampler's method as described by Blake and Hartge (1986).
Soil particle density was determined by using pycnometer method.
Soil porosity was calculated by using the following formula; Bouyoucos hydrometer method was used to determine percentage of sand, silt and clay. Textural class was determined by following the International Textural Triangle (Ryan et al. 2001). Infiltration rate was measured with a double ring infiltrometer. Soil saturated hydraulic conductivity was measured by Guelph permeameter (Model 2800 KI), taking three steadystate readings. The K fs was then calculated from the following formula; where R 1 and R 2 are the steady-state rates of water fall (cm S −1 ) in the reservoir at the first (h 1 ) and the second head (h 2 ) of water (cm), respectively, and X (35.5 cm 2 ) is the reservoir constant which is related to the cross-sectional area of the combined reservoir (cm 2 ).
Nitrogen was determined by Kjeldahl method. Soil organic carbon was determined at up to 1 m depths following the method described by Ryan et al. (2001).

Bulk density = Mass of oven dried soil∕
Volume of soil including pore spaces Soil particle density = Mass of oven dried soil∕Volume of soil excluding pore spaces where A = Total Volume of the extract (mL), V = Volume of extract used for measurement (3 mL), Wt = Weight of air-dry soil (g).
Total plant biomass, spike length, number of spikelets spike −1 , number of grains spike −1 , number of tillers m −2 and grain yield were recorded at crop maturity from each experimental unit. A strip of square meter was taken from each experimental unit, and measurements were recorded. Plant height was recorded at maturity.
Harvest index is the ratio of grain weight to the total plant weight at harvest and was calculated (Sinclair 1998) as: where GW is grain weight (kg) and TPW is the total plant weight (kg).

Statistical analysis
The collected data were statistically analyzed by using analysis of variance (ANOVA) techniques according to RCBD. The means were compared by least significant difference %Oxidizable organic carbon Soil Organic Carbon concentration g kg = %Total organic carbon × 10 (LSD) test at P ≤ 0.05 (Steel et al. 1997). The software packages STATISTIX 8.1 were used for statistical analysis. Data were analyzed for mean effect using two tailed Student distribution (T Test).

Results
Plant height was highest in over irrigated wheat crop which was statistically similar the crop which was irrigated following farmer practice while minimum plant height was observed in case of deficit irrigation (Table 2). Maximum spike length results from over irrigation followed by farmer practice irrigation application, while deficit irrigation resulted in lowest spike length (Table 2). Over irrigation showed maximum number of spikelets per spike which was followed by farmer practice of irrigation application, whereas deficit irrigation application produced minimum number of spikelets per spike of wheat crop (Table 2). Lowest number of grains per spike was recorded from deficit irrigation application, while farmer practice of irrigation application produced maximum number of grains per spike (Table 2). Farmer practice of irrigation resulted in maximum a productive tiller per unit area which was followed by over irrigation, however: deficit irrigation produced minimum number of productive tillers (Table 3). Maximum 1000-grain weight was recorded in wheat crop which was irrigated according to farmer practice, while minimum 1000grain weight was observed in deficit irrigation application (Table 3). Biological yield of wheat crop was significantly influenced by different irrigation applications. Maximum biological yield was recorded in wheat crop which was with over irrigation which was also similar to farmer practice of irrigation application, whereas deficit irrigation produced minimum total biomass of wheat crop (Table 3). Maximum grain yield was recorded in farmer practice of irrigation application which was followed by over irrigation; however, farmer practice and over irrigation resulted in similar grain yield. Whereas: minimum grain yield was noted from deficit irrigation application (Table 3). Similarly, farmer practice of irrigation application resulted in maximum harvest index of wheat crop which was similar to over irrigation, however: deficit irrigation application to wheat crop produced lowest harvest index (Table 3). There was a significant difference among various levels of irrigation applications on physical and chemical properties of soil. After harvesting of wheat crop soil analysis at soil depths of 0-25, 25-50, 50-75 and 75-100 cm indicated that deficit irrigation application resulted in maximum soil bulk density at all depths, while lower soil bulk density was recorded from over irrigation and farmer practice of irrigation application (Table 4). Soil particle density was lowest in over irrigated wheat crop which was followed by the soil irrigated according to farmer practice of irrigation application while deficit irrigation resulted in maximum soil particle density (Table 4). After harvesting of wheat crop, soil infiltration rate was recorded maximum from over irrigated soil which was followed by the soil irrigated according to farmer practice of irrigation. Minimum soil infiltration rate was observed in the soil which was provided with deficit irrigation (Table 4). Different irrigation levels had significant influence on saturated soil hydraulic conductivity and highest soil saturated hydraulic conductivity was recorded from over irrigated soil which was similar to that of farmer practice of irrigation application, whereas deficit irrigation showed minimum soil saturated hydraulic conductivity after harvesting of wheat crop (Table 4). Maximum soil porosity was observed in over irrigated soil which was similar to farmer practice of irrigation application while deficit irrigation resulted in minimum soil porosity at wheat crop harvest (Table 4). Soil analysis indicated that after harvesting of wheat crop nitrate concentration was significantly different at soil depths of 0-25, 25-50, 50-75 and 75-100 cm in over irrigated soil, soil which was irrigated according to farmer practice of irrigation application and deficit irrigation application (Table 5). Maximum nitrate concentration at soil depth of 0-25 cm was recorded from over irrigated soil which was similar to farmer practice of irrigation, whereas deficit irrigation application in wheat resulted in minimum nitrate concentration at 0-25 cm soil depth (Table 5). Similarly, at soil depths of 25-50, 50-75 and 75-100 cm similar trend was recorded in nitrate concentration (Table 5). Soil organic carbon was maximum at soil depths of 0-25, 25-50, 50-75 and 75-100 cm in over irrigated soil which was followed by the soil irrigated according to farmer practice of irrigation application, whereas minimum soil organic carbon was recorded in the soil provided with deficit irrigation in growing season of wheat crop (Table 6).

Discussion
Outcomes of the current study depicted that maximum plant height was produced over irrigation which was statistically at par with farmer practice (Table 2). Plant height was favored as irrigation was provided to wheat crop at all critical stages of crop growth Qiu et al. (2008). The results of current study are comparable with that of Li et al. (2005) who observed that maximum number of fertile tillers were recorded when wheat crop was irrigated at crucial stages of growth, while minimum productive tillers were observed when crop was irrigated at longer intervals. Increase in plant height might be due to increase in fertilizer rates to crop that boosted vegetative growth of plants by giving proper nutrients. According to Niaz et al. (2004), increase in the rate of irrigation levels lead to higher straw yield. Higher irrigation rates result in maximum spike length (Kabir et al. 2009).
The results depict that higher number of grains per spike attained when crop is irrigated at shorter intervals, while application of irrigation at longer intervals produced less number of grains per spike. Inadequate moisture availability at mid-season resulted in less number of grains per spike while at terminal stages of plant growth higher temperature along with water scarcity negatively influence grain weight in wheat crop (Messmer et al. 2009). According to Singh and Sharma (2001), different growth parameters like plant height, no. of tillers and dry weight increased with the increased rate of N to wheat. Our results are in accordance with Kabir et al. (2009) who reported increase in the number of fertile tillers with increased no. of irrigations. Niaz et al. (2004) demonstrated an experiment in which he compared the HI of maize crops obtained by using irrigation. He concluded similar results as in our study that there was non-significant difference between HI of maize crops obtained by application of irrigation. Hossain et al. (2004) recorded that bulk density increased significantly with soil depth, while effect of nitrogen was non-significant. Findings related to infiltration rate are in line with those of Iqbal et al. (2012). Similar trend of soil saturated hydraulic conductivity was observed by Iqbal et al. (2012). Sadej and Przekwas (2008) also observed increase in total nitrogen content with increasing N rates in the uppermost soil layer and soil nitrogen content decreased with soil depth. The increase in soil organic carbon (SOC) due to fertilization strategies could be due attributed to higher root biomass accumulated which might have added to the SOC particularly as indicated in the lower layers. Same trend was observed by Rasool et al. (2008) who concluded that balance amount of synthetic fertilizer can enhance SOC concentration up to 21%. Similarly, Sharma et al. (2002) reported increase in SOC due to inorganic fertilization. This was due to C addition through the roots and crop residues (Prakash et al. 2002) over the control plots. In another study, Jagadamma et al. (2007) observed SOC pool in 0-30 cm depth that ranged from 68.4 Mg ha −1 for N 0 to 75.8 Mg ha −1 for N 4 .

Conclusion
Outcomes of current experimentation depicted that number of leaves per plant, plant height and grain yield of the wheat were increased with increasing irrigation rates. Treatment I 2 produced maximum number of grains, 1000grain weight, total biomass and grain yield. Optimum level of irrigation enhanced the uptake of P and K while inappropriate application increased leaching losses. Bulk density and porosity of the soil remained unchanged under different treatment.
Acknowledgements This manuscript is extracted from thesis of M.Sc. (Hons.) Soil Science. I acknowledge the Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad, Pakistan for providing the resources including field and laboratory to conduct the research experiment.
Funding This research did not receive any specific funding.

Compliance with ethical standards
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