dentification and characterization of Mesorhizobial communities associated with chickpea crop in Thal region of Pakistan

By Asad Ullah

dentification and characterization of Mesorhizobial communities associated with chickpea crop in Thal region of Pakistan

Abstract

Chickpea (Cicer arietinum L.) being the 2nd most important short durational leguminous crop is grown throughout rain-fed areas of the world including Pakistan. In Pakistan, almost 1028.90 thousand hectares of land (4.3 % of the total cultivated area) is under chickpea cultivation with Punjab province contributing about 80% of the total produce. Under current scenario, output yield ratio from one hectare is less than 0.4 tons due to unavailability of nitrogenous sources and soil infertility. The only source of nitrogen in arid areas for chickpea is biological nitrogen fixation process through symbiotic relationship of Mesorhizobial communities. Hence in this study, to ensure the beneficial aspects of these communities towards nitrogen fixation through nodule formation, molecular characterization of the local Mesorhizobium  associated with chickpea roots was studied using 16s rDNA sequencing of the cultivable microflora. From 5 selected districts (Bhakkar, Miawali, Khushab, Jhang and Faisalabad) of Thal region, Twelve hundred and ten bacterial isolates were collected. 5 isolates were nominated for further molecular studies. The results depicted that out of these five selected isolates, four were assigned to genus Mesorhizobium based on 16S rDNA sequence analysis. The identity of isolates then were verified via BLAST method that depicted 80% to 90% similarities. Generally, this study has provided sustainable gateways for better understanding of Mesorhizobial association with chickpea and nitrogen fixation process at molecular level in Pakistan.

Introduction

Chickpea (Cicer arietinum) is cultivated in 5 continents over 40 countries including India, China, Pakistan, Russia, Egypt, Greece, North Africa, Italy and Rumania. It is an oldest legume crop in Asia and Europe which preferably originated from Mediterranean or Himalayas region. Chickpea crop produced highly nutritive and protein rich (almost 21%) seeds that consumed in all over the World (Nawab et al., 2008) as green vegetable, fried and boiled seeds, and flour. The very popular chickpea dish in Pakistan is “Dal Chana”. The leading producers of chickpeas are developing countries which contribute 95% of overall production. Considering Pakistan, Chickpea alone contributes 80% of country’s pulse production with Thal region of Punjab as a leading producer. Being the 3rd largest producer, Pakistan is facing several challenges which includes international market policies, lack of high yielding cultivars, unsubstantiated extension facilities, soil barrenness, area reduction, environmental variation and pathological problems.

Considering the growth habit, it is a self-pollinated, small, herbaceous much branched crop of sixty centimeter height. The leaves of chickpea crop are pinnately compound, light green to dark green in color generally with one terminal leaflet and covered with granular hairs. Pinnate leaves have small 9-15 leaflet of various size, shape and color with serrated edges and red margins. Flowering process begins over early on the second day and anthesis starts from 9 AM- 10 AM and continues till 3 PM. The color of the flowers varies from white to shade of blue or pink. The pods usually are two cm long and contains two seeds per pod while a single plant have approximately 50-150 pods.

Chickpea plant has a developed nodule associated root system including central tap root, adventitious roots and extensive network of root hairs that spread out in all direction of soil’s upper layer. These nodules are filled up with Mesorhizobial bacteria that fix nitrogen form atmosphere on continual basis and transforms it into an energy rich source called ammonia.

On the basis of seed morphology and color, chickpea is divided in to two basic types, The Desi type: in which mostly the seeds are small, rough and angular in shape. The seed coat of desi type is thick and colorful while the common colors are black, green, yellow and brown. The flower of desi type are mostly of pink color due to the presence of anthocyanin pigmentation on the stem. In Pakistan, more than 70-80% of cultivated chickpea is of Desi type. Dal and flour are always made from this type. The Kabuli type chickpea are characterized by beige or white seed colored seed with ram’s head shape, white flower, thin seed coat, smooth seed surface and the absence of anthocyanin pigmentation. Kabuli type as compare to desi type has higher percentage of sucrose and lower percentage of fiber. The seed of Kabuli type generally large in size and due to this it gets higher market price as compare to desi type.

Usually chickpeas are grown as a rainfed cool weather crop or as dry climate crop in semi-arid regions. Desi type chickpeas are more tolerant to dry season than Kabuli type due to small gain size. Excessive rainfall, frost, drought, hailstorm and nutrient deficiency may led to the severe crop damage. Relative humidity for optimum seed setting must be between 21-41%.

Due to miraculous potential and drought resistant abilities, chickpea crop can be grown in wide agro-ecological zones and soil types. In Pakistan, chickpeas are generally grown on clay loam and sandy loam soils but the most suitable soil is the sandy loam soil of Punjab regions i.e. Thal and Cholistan. The soil excessive in soluble salts, neutral in reaction or having pH greather than 8.5 is not suitable for chickpea crop.

In leguminous crops nitrogen fixation mostly depends upon the symbiotic relationship of plants and Mesorhizobial communities. These communities are the group of gram negative bacteria that facilitate plant in nodule formation and atmospheric nitrogen fixation.(Babic et.al., 2008).

The purpose of this study were to inspect the indigenous diversity of  Mesorhizobial species associated with chickpea crop in Thal region through PCR based 16S-rDNA sequence analysis, to determine the  relationship among these isolates, to understand the symbiotic relationship of rhizobia with host plant and finally to peruse scientist for development of proficient  bio-inoculant.

Before studying different biological, physical and molecular characteristics of rhizobia associated with chickpea, it is requires to isolate them first. The traditional methods for isolation of Rhizobia are much laborious and time consuming. Isolation actually involves treating each and every nodule separately while sterilizing and disrupting the nodule surface to release bacteria. Nodule sterilization is done by either washing nodules in distilled water or dipping in 70% of ethanol solution. While nodule disruption is accomplished through cutting, crushing or stabbing the nodules.

Rhizobia have symbiotic relationship with legumes and every specie has altered host range of plants with numerous metabolic proficiencies (Margaret et al., 2011). Under field conditions, various parameters for rhizobial symbiotic efficiencies are anticipated but the most important factors in chickpea symbiosis were: (i) the occupancy of nodules, that directly relates with endurance competitiveness among the bacterial community (ben Romdhane et al., 2007) and (ii) the nitrogen fixation affectivity, which strengthen the plant growth and yield (Ben Romdhane et at., 2008).

In recent years, 16S rDNA sequence analysis emerged as one of the divergent and important method for bacterial taxonomy and phylogenetic studies (Weisburg et at., 1991) but due to high sequence conservation

Sequence analysis of 16S ribosomal RNA (rRNA) has emerged as one of the most important methods in taxonomy and phylogenic analysis of bacteria (Weisburg et al. 1991).

However, due to the high sequence conservation, this techniques is insufficient to distinguish the strain of several species together.

In Pakistan farmers from “Barani tract/arid areas” in Thal and Cholistan desert areas (Bhakkar, Layyah, Muzafar garh, Jhang, Mianwali, Khoshab; scarce rainfall, hot and dry conditions with low nutrient availability through sandy soils) do not have the sufficient economical resources to buy farm inputs. In Pakistan, almost 479.5 thousand tone of chickpea (average yield of 466 kg/ha) is produced from 1028.90 thousand hectares (4.3 % of the total cultivated area), with Punjab province contributing about 80 percent of the total produce coming from Cholistan and Thal deseret areas. In these areas more than 90% agro-based income is generated from chickpea cultivation of Kabuli and Desi varieties. In these areas chickpea is sown on far separated sand dunes literally called “Tibbas” on residual soil moisture with no farm inputs through traditional agriculture systems. The total output yield is totally dependent upon ecological factors and farmers have very limited choices in terms of provision of better adapted germplasm, disease diagnostics, fertilizer and other farm inputs. Under such circumstances, for chickpea, the symbiotic nitrogen fixation by rhizobia species becomes crucial for supplying available nitrogen (Kaneko et al., 2000), salt and drought tolerance, general plant vigor and higher yields, and hence can be a target for developing low cost cropping solutions for sustainable chickpea production. Therefore, an immediate characterization of rhizobial strains associated with local chickpea germplasm is deemed necessary, for which the present research work is designed with following objective:

Review of Literature

Chickpea importance

Chickpea being the member of genus Cicer, family Fabaceae and subfamily papilionaceae is world’s 2nd largest leguminous crop which has been originated in southeastern Turkey (Ladizinsky, 1975). Genetically, chickpea is a self-pollinated, annual diploid crop having chromosome number 2n=16 (Rajput et al.). The name Cicer is derived from the Greek word “kirkus” which means the force. According to Duschak (1871), this word also have some roots in Hebrew word “kirkes” which means round in shape. While the 2nd part arietinum is derived from a Greek word “krios” which means ram, it is an insinuation to the shape of chickpea seed which actually resembles with the head of a ram (Van der Maesen 1987). The wild species of chickpea are found in all over the world but abundant in Pakistan, Turkey, Central Asia, Iran and Afghanistan (Duke, 1981).

            In central Asia, chcickpea is normally accredited as gram and is the most vital Rabi pulse crop of Pakistan. More than 10000 families in rainfed areas of Pakistan are dependent on chickpea crop for their survival.  In Pakistan, especially in Punjab, chickpea is progressively grown in Bhakkar, Mia wali, Khushab, Layyah, Jhang, Chakwal, Attock and Jhelum (Haqqani et al., 2000).

A lot of work has been done related to the genetic diversity of chickpea associated Mesorhizobia, effect of Mesorhizobial communities on nodulation or nitrogen fixation and production/yield variation of chickpea crop (Jeena et al., 2005) associated with above mentioned factors throughout the world. But the facts about genetic studies of Mesorhizobia and nodulation related to agro-climatic conditions in Pakistan are hardly accessible in literature. Being a main cash crop of arid areas, chickpea prefers to grow on sandy soil and its production completely depends upon judicious rainfall throughout growing season. Due to rainfall uncertainty, low attentive behavior towards research and development and abrupt changes in climatic conditions, it is difficult for the farmers to decide whether the input usability would be economical or not. Mesorhizobial community having symbiotic relationship with the roots of leguminous plant for root nodulation is collectively known as rhizobia. Nearly 13 genera of Proteobacteria are associated with nodule formation in chickpeas (Dudeja and Narula, 2008; Dudeja et al., 2012).

Phenotypic and genotypic characterization of chickpea Mesorhizobia

Phenotypic characterization of bacterial population includes morphological, biochemical, physiological and symbiotic compatibility of crop and its associated Mesorhizobia. For numerical taxonomic purposes, phenotypic based substrate tests are commonly used (Gao et al., 1994) which provides ecological information when organism use that substrate). 16S rDNA gene sequencing used for genotyping by various methods including polymerase chain reaction (PCR) (Boudewijns et al., 2006) and restriction fragment length polymorphism (RLFP) (Laguerre et al., 1994).

Genotypic characterization for nodulation and the impact of Mesorhizobia on nodulation

Significant nodulation variations and high heritability among different chickpea genotypes has been reported (Yadav et al., 2004; Gillani et al., 2005; Mensah and Olukoya, 2007). In 1992, it was reported that the frequency for Nod-plant fluctuated from 120 to 490 per million under field condition (Rupela, 1992). It was proved in 20th century that there is a significant correlation between nodulation and seed yield. (Corbin et al., 1977). It was studies that nitrogen requirement for plant or grains are mainly fulfilled by nodule fixated nitrogen rather than any other source i.e. nitrogen fertilizers/ supplements (Hungria and Neves, 1987). Highly specified symbiotic relationship among chickpea plant and Mesorhizobial communities is necessary for nodule formation and nitrogen fixation. Any ailment with Mesorhizobial communities such as poor growth, low population number, existence of inappropriate diversity or ineffectiveness of strains may led to nodulation and nitrogen fixation problems.

Different microsatellite markers have been used for the investigation of different chickpea genotypes and one of them is Simple Sequence Repeat (microsatellite) which is considered as efficient tool for assessment of genetic variability pattern (Winter et al., 1999) while for the Mesorhizobial species 16srDNA has been a successful technique.

Materials and methods

Survey and sample collection

Surveys was conducted during chickpea growing season in five districts of Thal region including Faisalabad (as model location), Jhang Miawali, Khushab and Bhakkar to collect samples of chickpea roots associated nodules.

Random sampling was performed in chickpea field and dunes of Thal region including Faisalabad. Twenty locations were selected from each of the five district and in each location, five sites were again selected and desired samples were collected. Major sampling sites from all districts was 7/1 Thal Janboobi, 7/2 Thal Janoobi, Athara Hazari, Nawan kot, Rudu Sultan, Jabboana, Chely Wala, Chenny wala, Burji Wala, Hedarabad Thal, Azri bhakkar, NIBGI Trail Fields, Mankera, Kalorkot, Darya khan, Khansar, Jhok Mehr Shah, Jhang Rd. Kaimar, Dera Dhona Wala, Pelo venus, Noor pur thal. Sha Hussan, Roda, Arsalpur, Chandni Chowk, Nawan Sago, Ali khel, Jhumtaan Wala, and hernoli   On the whole 100 samples were collected from each district and 500 samples from all districts.

Sample Harvesting Procedure

Roots of plants were harvested from field by digging up the area below the crown of plant using shovel. Soil was removed carefully from roots so that nodules remained fresh (washed nodules remain fresh up to 5 days) and then roots having nodule were placed in plastic bags.

Nodule separation and preservation

Nodules from all samples were separated, counted and preserved in sterilized 96 well plates containing 2-5 beads of silica gel. The filled plates were stored in refrigerator at -4◦C for further microbial studies such as for rhizobial isolation.

Isolation of Rhizobia from Nodules
Collected nodules associated with Mesorhizobial communities were isolated using different culture media including Nutrient Agar and Yeast Extract Mannitol (YEM) Agar with . Each nodule was surface sterilized with 70% ethanol for 30 seconds and rinsed twice with distilled water and then nodules were crushed into pieces. Crushed nodules were plated on the patriplates having rhizobia specific media (YEM). For crushing and plating, needles and scissors were used that were sterilized by dipping in methylated spirit and flaming several time. All the petriplates were incubated at 27◦C ± 1 for 4-7 days and daily observations were recorded.

Purification of Rhizobial Isolates

Rhizobial isolates were purified on YEMA (Yeast Extract Mannitol Agar) containing 0.0025% Congo red. YEMA is highly specific and routinely used media for rhizobia isolation and purification (Vincent, 1970).

YEMA was prepared by adding mannitol 10g; agar 15g; potassium hypochlorite 0.5g; Yeast extract 0.4g; Magnesium sulphate 0.2g;  NaCl 0.2g; Congo red 0.025g and 1 liter of distilled water in conical flask and then covered with cotton plug. Then the mixture was placed on stirrer for homogenization. After that the mixture was autoclaved at 121◦C temperature and 15 psi pressure for 15 minutes then prepared medium was placed at optimum conditions for cooling. Finally, medium was poured in Petri plates in laminar flow chamber. When the media was solidified, a single colony from each nodule extract was taken directly and streaked on congo red containing YEMA plates. Subsequent streaking (3 to 4) were done to obtain purified isolates.

Culture Preservation

Culture of isolated rhizobial isolates will be prepared in YEM broth medium through following steps.

Inoculum Preparation

5ml of sterile YEM broth medium was poured in each test tube then isolated subculture colonies was inoculated separately in each YEM tube via sterilized loop. Controlled was also maintained which contained only YEM liquid media for comparison. Finally, inoculated tubes were placed on shaker at 28◦C for 12-16 hrs for thorough mixing and better colonial growth.

Preparation of Glycerol stock

50% glycerol stock was prepared by mixing 50 g glycerol and add distilled water up to 100 ml then the mixture was autoclaved. After autoclaving, 0.5-0.75 ml culture was taken with equal volume of 50% glycerol in Ephondrof tubes.finally, Eppendrof tubes was placed at – 40◦C.

DNA extraction from isolates

Rhizobial isolates were grown in YEM broth and incubated at 27±1°C for overnight under shaking at 220 rpm. About 1.5 ml of prepared culture was taken in a micrcentrifule tube, spin for 7 minutes and supernatant was discarded. Now add 400 μL TE buffer to re-suspend the pallet, add 10 μL of 20% sodium dodecyl sulphate and vortex the mixture. Now add 100 μL of proteinase K, vortex the mixture complelety and incubate it at 37°C for 1hour. Now add 420μL phenol chloroform and vortex the mixture. Now centrifuge the suspension at 14ooo rpm for 15 minutes. Now carefully take the supernatant in to new eppondrof tube and add 15 μL of 3 molar sodium acetate and mix it well. Then add 570 μL of isopropanol and again mix it gently. Incubate the resultant at -80°C for 15 minutes and then again centrifuge at 14000 rmp for 15 minutes to get the pallet in the mixute. Now remove the supernatant by decanting the eppondrof tube and wash the pallet by using 200 μL of 70% ethanol. Again centrifuge at 14000 rpm for 1 minute and remove the liquid with the help of micropipette. In the last, air dry the pallet at 37°C for 15 minutes. Now add 25 microliter of TE buffer to re-suspend the pellet and store it at -20 °C. for PCR reactions

PCR based 16S rDNA sequencing

The rDNA of isolated rhizobial strains was amplified by using standard protocol described by Sambrook et al., 2001. PCR was performed in thermal cycler by using Universal Primers (Forward primer: fD15 CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3, Reverse primer: Rd15 CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC-3) (Wen et al., 1999). The Gel (1%) electrophoresis of the PCR product will be done to find out the band size.

Phylogenetic Relationship studies for Mesorhizobial communities

Phylogenetic studies for isolated Mesorhizobial strains will be conducted by using MEGA-6 phylogenetic software and the expected product fragment size will be 1500bp.

Phylogenetic relationship will be find out by BLASTING these 1 6S rDNA sequences of rhizobial bacteria on NCBI.

PCR amplification and Phylogenetic investigations: The standard protocols as discussed by Sambrook et al., 2001 will be followed for amplification of genomic DNA. PCR will be performed in thermal cycler. Specie specific forward primer and reverse primer will be used for PCR amplifications. PCR master mix will constitute of 18µLwater, buffer containing MgCl2 2.5µL, dNTP (2.5 mM) 0.5µL, forward Primer (1 µmol/µL) 1µL, reverse primer (1 µmol/µL) 1 µL, DNA polymerase (2U/µL) 1µL, template (25 ng/µL) 1µL. The final PCR products will be resolved in 1.0% agarose. The gel will be stained with ethidium bromide and photographed on UV trans-illuminator. Data analysis and interpretation of results will be done by comparison of banding pattern generated from each strain. The tree plot will be constructed by UPGMA (Un-weighed pair group arithmetic average) to analyze the chronological separation of isolates. Preliminary phylogenetic analyses were performed using the neighbour-joining method (Saitou and Nei, 1987) using the MUST package (Philippe, 1993) to facilitate the selection of representative sequences (Abram, 2014).

Total collected samples

District  Name

Total sample

Nodule Plant

Jhang

100

81

Bhakkar

100

93

Mianwali

100

76

Khushab

100

79

Faisalabad

100

96

Total

500

425

 

No. of nodules separated from all Districts

District  Name

Total sample

Nodule Plant

 Total nodules

Jhang

100

81

1470

Bhakkar

100

93

2104

Mianwali

100

76

1105

Khushab

100

79

1143

Faisalabad

100

96

2215

Total

500

425

8037

Total Isolates

District  Name

Total sample

 Total Isolates

Jhang

100

283

Bhakkar

100

248

Mianwali

100

213

Khushab

100

198

Faisalabad

100

268

Total

500

1210

 

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