Specific RT-PCR assays for the detection of Trichoderma harzianum (Th azad) in rhizopsperic soil sample of Uttar Pradesh India.
In recent years, several PCR-based molecular techniques have been used to detect and discriminate among microorganisms. Sequence analysis of the internal transcribed spacer region 1 (ITS1) of the ribosomal DNA has been helpful in the neotypification, description, and characterization of species in the genus Trichoderma [Gams, W, et al. (1998), Shahid M et al. (2013a, 2014f), Grondona, I et al. (1997), Hermosa, MR et al. (2000), Lieckfeldt, E, et al. (1999), Muthumeenakshi, S. et al. (1994), Ospina-Giraldo, MD et al. (1999)]. In addition, the analysis of DNA sequences from multiple genetic loci has been used to establish the phylogenetic relationship of the species within Trichoderma [Chaverri, P. et al. (2003), Kullnig-Gradinger, C. et al. (2002)]. Another useful method for the identification of Trichoderma strains is the randomly amplified polymorphic DNA (RAPD) technique [Zimand, G et al. (1984), Shahid M et al. (2013b, 2014g)]. In a previous study, we identified RAPD fragments that were able to discriminate between different biocontrol agents of Trichoderma spp. [Hermosa, MR et al. (2001)]. By converting a RAPD- derived fragment, amplified from strain T. atroviride 11, into a sequence-characterized amplified region (SCAR) we showed that this marker was specific to T atroviride 11. The generation of a SCAR marker for this strain is of particular interest because T. atroviride 11 has been shown to be an efficient biocontrol agent against phy- topathogenic fungi such as Phomabetae, Rhizoctonia so- lani, Sclerotinia sclerotiorum, and Polymyxa betae, the vector of the sugar beet rhizomania disease virus [Campos, T et al. (2001), Grondona, I, et al. (2001), Grondona, I. et al. (1992)].
Although conventional PCR has become an attractive tool for the detection of specific microorganisms in microbial systems, this technique does not allow accurate quantification of DNA because of the variability in the efficiency of amplification between PCR reactions [Raeymaekers, L (1998)]. This limiting factor has been overcome by the emergence of new techniques, capable of quantifying nucleic acids in vitro. Real-time PCR is based on the use of TaqMan probes or SYBR Green I dyes. Both systems measure the intensity of a fluorescent signal proportional to the quantity of DNA generated during the PCR amplifica- tion. Quantitative real-time PCR is widely used in medicine for the diagnosis of clinical viruses [Kimura, H. et al. (1999).], bacteria [Hein, I, et al. (2001), Ke, D, et al. (2000)], protozoa [Lin et al. (2000)], and fungi [Loeffler, J. et al. (2000)]. Although it is used in plant pathology studies [Schena, L. et al. (2002) ], there are few references regarding its application to quantifying fungi from soil [Filion, M. et al. (2002)].
The aim of the present study was to develop specific PCR-based markers to detect T. harzianum Th azad in soil. Also, two primers, QTh azadf and QTh azadr, designed from the 837-bp SCAR marker, were used to quantify fungal DNA of this strain extracted from soil samples using real-time PCR.
Fungal Strains, Growth Conditions, and DNA Extraction
The 49 Trichoderma spp. were used in this study are shown in Table 1. Each strain was cultured on Potato Dextrose Agar medium (PDA, Himedia) and stored at -80[degrees]C in 10% glycerol. Petri dishes containing PDA medium were inoculated with a 0.5-cm diameter agar plug, cut from the growing edge of a 4-day colony for each strain, and grown in the dark at 25 C. Spores were harvested after 6 or 7 days, by adding 2 mL of water to the plates and scraping the culture with a rubber spatula. The suspensions were filtered through a double layer of cheese- cloth to separate large mycelia fragments from conidia. Spore concentrations were adjusted to 1 x [10.sup.9] conidia mL). Mycelia, for each strain, were obtained by inoculating flasks containing 100 mL of Potato Dextrose Broth (PDB, Himedia Laboratories) and growing the cultures at 25[degrees]C for 48h on orbital shaker (120 rpm). Mycelia were harvested and washed with sterile distilled water, frozen, and lyophilized. Total DNA was extracted from 50 mg of freeze-dried mycelia [Shahid M. et al. 2014a, Mondejar R L et al (2010), Raeder, U. et al. (1985)].
DNA extracted from 250 mg of soil was obtained using the Fast DNA SPIN (for soil) kit (according to manufacturer's instructions, with a modification in the lysis step. Lysis was carried out using the solution provided in the kit. However, the samples were incubated at 65 C and vortexed every 5 min for 30 min. The DNA extracted from each sample was resuspended in 100 1L of DES buffer accompanying the kit.
Screening Trichoderma Strains for SCAR Markers
SCAR A1 (5 GGAAGCTTGGCGTTTATTGTA CAAAG-3) and SCAR A1c (5-GGAAGCTTGGGTATT GAGCTGGGCCT-3) primers, designed from a previous study involving T. atroviride 11 [Hermosa, MR. et al. (2001)], were used to test the amplification of DNA from 50 strains of Trichoderma spp. Each PCR reaction contained 50 ng of fungal DNA (Table 1), 2.5 U of DNA Taq polymerase, 1 * DNA polymerase buffer , 0.2 mM of each deoxynucleoside triphosphate, 1.5 mM of MgCl2, and 0.32 lM of each primer in a total volume of 50 lL. PCR conditions included an initial denaturation of 5 min at 940[degrees]C, followed by 35 cycles of 1 min at 94[degrees]C, 1 min 30 s at 68[degrees]C, and 2 min at 72[degrees]C, with a final extension step of 72[degrees]C for 7 min, using a DNA PTC-100 thermal cycler. PCR products were analysed by electrophoresis in a 1.2% agarose gel in 1 * TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA [pH 8]), stained with ethidium bromide. The molecular size marker was / X174- HaeIII (Promega, Madison, WI, USA).
Generation of a SCAR Marker for T. harzianum Th azad
A 1.5-kb PCR fragment was excised from an agarose gel and the DNA was purified with the GENE CLEAN kit according to the manufacture's specifications. The fragment was cloned into the pGEM-T Easy vector (Promega) with T4 DNA Ligase [Vieira PM et al (2013), Sambrook, J. et al. (1989)]. Plasmid DNA was extracted using a miniplasmid preparation protocol [Hanahan, D (1985)]. The 1.5-kb fragment was sequenced using fluorescent terminators on an AB RT PCR.
Their identification is important in developing a potential strain for further analysis. These isolate belongs to 8 different species of Trichoderma genus viz. Trichoderma viride (30 strains), Trichoderma harzianum, Trichoderma asperellum, Trichoderma longibrachiatum (7 strains), Trichoderma atroviride (8 strains), Trichoderma koningii, Trichoderma virens and Trichoderma reesei collected from rhizospheric soil of chickpea, pigeonpea and lentil crop. They are finally deposited to culture bank NBAIM, Mau and allotted with a unique NBAIM Accession number.
Real-Time PCR Assays. One primer set, QTh azadf (5-TGGCGTTGAATTGAGTTTGTGT3) and QTh azadr (5-CCCTCCGTA TGGGTTT TAAGGT-3) , was designed using the Primer Express software program version 2.0.0 (Applied Biosystems). These oligonucleotides, designed from the 837-bp SCAR marker, flanked a 72-bp DNA fragment specific for identifying the strain T. harzianum Th azad. The specificity of this primer set was tested against DNA from all 50 Trichoderma spp. strains, using a conventional PCR machine, and on annealing temperature of 59[degrees]C for the reactions.
The QTh azadr and QTh azadf primers were used in real time-PCR experiments with 6-carboxyfluorescein (6-FAM) as the reporter fluorochrome & 6-carboxyte- tramethylrhodamine (TAMRA) as the quencher. The 20- lL master mixture contained 2 lL of undiluted DNA template from different sources, 10 lL of TaqMan Universal PCR Master Mix (Applied Biosystems), a 0.45-lM concentration of each primer, and 0.5 lM of a TaqMan fluorescent DNA probe (Applied Biosystems). Thermal cycler conditions were as follows: 50 C for 2 min and 95 C for 10 min, and 40 cycles of PCR amplification at 95 C for 15 s and 59 C for 30 s.
A standard curve, based on threshold cycles (Ct), was created using plasmid DNA containing the 837-bp SCAR marker, and the QTh azadf/QTh azadr primer pair. Plasmid DNA amounts, quantified by spectrophotometry and serially diluted, were 200 pg, 20 pg, 2 pg, 200 fg, 20 fg, and 2 fg. Sterile water was used as a negative control to replace template DNA in PCR reactions (NTC). Ct values were calculated by the ABI Prism 7000 SDS software program (Applied Biosystems) to indicate significant fluorescence signals rising above background during the early cycles of the exponentially growing phase of the PCR amplification process. The standard curve was obtained by plotting the Ct value, defined by the crossing cycle number, versus the logarithm of the quantity of the different plasmid DNA samples. Two microlitre of undiluted DNA from the A, B, C was included as template in the real-time PCR reactions Two microlitre of genomic DNA was also included as positive control. The amount of template DNA was calculated by interpolating the cycle threshold with the standard curve, determined by the ABI Prism 7000 SDS software program, and by the application of a correction factor. DNA quantities were corrected by dividing by 35, a figure that represented the ratio between the 110-bp target fragment and the total size of the plasmid DNA used to generate the standard curve. All reactions were carried out in triplicate.
SCAR Marker Generation for T. harzianum Th azad
Two PCR products were amplified using SCAR A1 and SCAR A1c primers: one 990-bp fragment corresponding to strain T. atroviride 11 and one 1.5-kb fragment corre- sponding to T harzianum Th azad . The 990-bp fragment was shown to be diagnostic of T atroviride in work previously done using 17 strains of Trichoderma [Hermosa, MR. et al. (2001)]. Two primers, BR1 and BR2, were designed to the 1.5-kb fragment from T. harzianum Th azad and used to amplify DNA extracted from 49 strains of Trichoderma spp..Of these samples, only one 1.5 kb amplicon, specific to a sample containing only T. harzianum Th azad DNA, was amplified using an annealing temperature of 65[degrees]C. No PCR products were detected in any of the other 48 PCR reactions.
Detecting Trichoderma in Inoculated Soils
Sample A, a sterile soil artificially inoculated with a mix of 49 Trichoderma spp. strains at a concentration of 109 spores per 10 g of soil, yielded 1.6 to 5.6 lg of DNA per g (w/w) of soil. No differences in the amount of DNA were observed in soil samples. The amount of DNA extracted from soil sample, a sterile soil inoculated only with T. harzianum Th azad at a concentration of 109 spores per 10 g of soil, was not detected in agarose gel.
BR1 and BR2 primers were used to screen the DNA from the soil samples (Table 1) to discriminate between samples with or without T. harzianum Th azad. The diagnostic 1.5 Kb PCR fragment was amplified in soil samples which included spores of T. harzianum Th azad. No PCR products were amplified in samples, where spores of T. harzianum Th azad were not added (Fig. 1). Identical results were observed when mycelia instead of spores of the Trichoderma spp. strains were used to inoculate the soils. The quantities of DNA extracted from soils inoculated with mycelia were 60% higher than the amount extracted using spores as the inoculant.
Screening and Quantification of Fungal DNA Using Real-Time PCR
The QTh azadf/QTh azadr primer pair was used in real-time PCR. A standard curve was created using 4-fold dilutions series of the plasmid containing the 1.5kb SCAR fragment and both primers. The threshold was set to a value of 0.50 with a baseline ranging from cycle 1 to 36. The standard curve showed a linear correlation between input DNA and cycle threshold (Fig. 2), with a correlation coefficient (r2) of 0.100754. Significant fluorescent signals were observed for reactions containing DNA extracted from sample , inoculated with 109 spores of T. harzianum Th azad per 10 g of soil (Table 1). Genomic DNA of T. harzianum Th azad, included as positive control, was also detected. No significant fluorescent signals, rising above background, were detected for sample, which did not contain T. harzianum Th azad strain. In addition, no fluorescence was observed for sample, a nonsterile and artificially inoculated soil, which included 49 Trichoderma spp. strains distinct from T. harzianum Th azad. Thus, indicating that primers QTh azadf and QTh azadr did not amplify the DNA from other microorganisms or Trichoderma spp. strains present in this soil. The absence of T. harzianum Th azad propagules in sample was confirmed by plating triplicates of 100 mg of soil on Trichoderma specific medium, TSM [Askew, DH. et al. (1993)]. For sample A, fluorescent signals were detected, but this result was not consistent within the three replicates done for each experiment and could not be considered as a real quantification.
Strains identification in situ is an important factor in the monitoring of microorganisms used in the field. In this study, we demonstrated the use of Sequence Characterized Amplified Region (SCAR) markers to detect genomic DNA from Trichoderma strains from soil. Two primers (SCAR A1 (5' -GGAAGCTTGG CGTTTATTGTACAAAG-3') and SCAR A2 (5'-GGAAGCTTGGGTAT TGAGCTGGGCCT-3') were tested against DNA of 49 isolates of Trichoderma spp. and a 1.5 kb fragment from T. harzianum Th azad, using annealing temperature of 68[degrees]C. These fragments showed no significant homology to any sequence deposited in the database.
The identification of organisms on the basis of DNA investigation requires the characterization of discrimi- nating DNA targets. In fact, this is especially important in the case of Trichoderma strains widely used in the bio- control of soil-borne plant-pathogens. Although T. harzianum is the species most frequently used in biological control, a search of the sequences from commercial strains revealed that T. asperellum, T. atroviride and T koningii were also common species used [Hermosa, MR. et al. (2000), Samuels, GJ (1996)]. Moreover, because of the difficulty of monitoring the biocontrol activity of a given strain in natural environments, the development of molecular characters is needed. A recent work describes the cotransformation of a T harzianum strain with green fluorescent protein and GUS genes [Bae, YS. et al. (2000)] to study its ability to colonize soil and sclerotia of a plant pathogenic fungus. These genes provide a valuable tool for the detection and monitoring of specific strains of T. harzianum released into the soil. However, the practical use of these transformants is difficult because of the public concern about genetic modified organisms in natural environments.
PCR has provided a reliable method for the identification and detection of microorganisms. Specific primers have been designed from ITS regions or rRNA genes of fungi such as Rosellinia necatrix [Schena, L. et al. (2002)] or Glomus intraradices [Filion, M. et al. (2003) ], and specific primers from the Fusarium solani f. sp. phaseoli translation elongation factor 1 alpha (tefl) gene [Filion, M. et al. (2003)] have permitted the identification of these fungi. Several studies have indicated that some Trichoderma species are closely related [Gams, W, et al. (1998), Kullnig-Gradinger et al. (2002)]. Divergence values of ITS1 and ITS2 regions, ranging from 1.2 to 6.5%, 0 to 5.2% and 1.9 to 14.6%, have been shown for three sections of this genus: Longibrachiatum, Trichoderma, and Pachybasium [Shahid M. et al. 2013c, 2014b, Kuhls, K. et al. (1997)]. Similarly, sequence analysis of ITS1 revealed T. atroviride, T. asperellum, and T. harzianum differ by only 0 to 3% [Hermosa, MR. et al. (2000)]. A DNA sequence data analysis of four genes (ITS regions, tef1 a, calmodulin, and a-actin) from T. harzianum produced 1.5, 20.9, 14.8, and 5.8% of informative-characters, respectively [Shahid M. et al. 2013b, 2014d]. However, taking into account the high taxonomic value of these genes, they are not useful enough for designing strain-specific primers for monitoring Trichoderma isolates.
T. harzianum Th azad is commonly used in basic biocontrol research [De la Cruz, J, et al. (1999)] and produces a variety of cell-wall-degrading enzymes in natural substrata [Benitez, T. et al. (1998)]. Because of its value as a research model, this strain was considered in the present study. In previous work, a strain-specific SCAR marker was developed to distinguish T. atroviride 11 from 42 Trichoderma spp. strains, including biocontrol agents, belonging to 13 species [Shahid M. et al. 2013a, 2014c, Hermosa, MR. et al. (2000)]. In the present study, when the SCAR A1 and SCAR A1c primers were tested under annealing conditions at 65[degrees]C, A 1.5 kb fragments was amplified with the DNA from T. harzianum Th azad. The 1.5-kb band amplified from T. harzianum Th azad DNA with SCAR A1 and SCAR A1c primers and sequence analysis provided no evidence of homology with other GenBank sequences.
The specificity of BR1 and BR2 was tested against the DNA isolated from 49 Trichoderma species, The primer pair, BR1 and BR2, amplified an 1.5 KB product with T. harzianum Th azad DNA. These results suggest that we have identified a suitable molecular marker and that the PCR-specific primers, BR1 and BR2, are useful for detecting T. harzianum Th azad. The amount of DNA recovered from a given soil is highly related to factors such as soil type or the method of extraction. In most cases, the recovered DNA from soil is not proportional to the number of inoculated microbial propagules until values are >106-107 spores per g of soil [Frostegard, A. et al. (1999)]. In real-time PCR experiments primers QTh azadf and QTh azad did permit the quantification of a target fragment, contained within the SCAR marker of T harzianum Th azad, from known amounts of spores artificially inoculated in soil samples. The sensitivity of the technique was shown by detecting quantities <0.057 fg of the 1.5kb target fragment of T. harzianum Th azad, based on the values of the standard curve. In other real-time experiments using the Taq-Man system, 0.77 fg of Mycobacterium avium DNA was detected in fecal samples [Fang et al. (2002)]. Our results showed a higher sensitivity (10 times greater) using a Taq-Man probe. In soil sample containing only T. harzianum Th azad spores, DNA was quantifiable and the results were reproducible. Fluorescent signals were observed around cycle 31. In the case of soil sample, inoculated with a mixture of 109 spores per 10 g of soil of the 49 strains, including T. harzianum Th azad, the results were not consistent or reproducible. This may be explained by the use of a lower annealing temperature, which was determined by the Primer Ex- press program when primers QTh azadf and QTh azadr were designed. In initial experiments, 65[degrees]C was the annealing temperature that allowed the specific amplification of the 1.5 kb SCAR marker of T. harzianum Th azad, using BR1 and BR2, tested using the DNA from 49 strains. The annealing temperature used in real-time PCR with primers Q Th azadr and QTh azadf primers was 59[degrees]C and the total size of the target fragment was 1.5kb. Perhaps under these conditions the primers were nonspecifically binding to sites within the DNA of the various strains included within the mixture and therefore were not able to result in a positive or reproducible fluorescent signal for soil sample, whereas this was not the case for sample containing only T. harzianum Th azad. Primers QTh azadf and QTh azadr were also tested using conventional PCR with the DNA from pure cultures of 49 Trichoderma spp. Only one amplicon was detected with T. harzianum Th azad genomic DNA, from spores, indicating the specificity of Q Th azadf and Q Th azad.
SCAR primers BR1 and BR2 were able to detect T. harzianum Th azad using conventional PCR. However, even though QTh azadf and QTh azadr were able to detect T.harzianum Th azad in both pure culture and artificially inoculated sterile soil, their specificity was not great enough to detect and quantify T.harzianum Th azad among a mixture of strains. These results suggest that the use of real-time PCR to quantify a specific strain within natural soil containing a complex mixture of microbes is not reproducible with these particular primers and conditions. However, this does not mean that this strategy would not be useful to quantify a given species using multicopy genes such as those from the rRNA cluster [Schena, L. et al. (2002)].
The authors are grateful for the financial support granted by the ICAR under the Niche Area of Excellence on Exploration and Exploitation of Trichoderma as a antagonists against soil born pathogen, running in Department of Plant Pathology, C.S. Azad University of Agriculture and Technology, Kanpur.
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Mohammad Shahid, Mukesh Srivastava, Sonika Pandey, Vipul Kumar, Anuradha Singh, Shubha Trivedi and Y.K. Srivastava
Biocontrol Laboratory, Department of Plant Pathology, CSA University of Agriculture & Technology, Kanpur--208002, India.
(Received: 09 September 2015; accepted: 01 November 2015)
* To whom all correspondence should be addressed. E-mail: email@example.com
Caption: Fig 1. Showing expression of T. harzianum Th azad DNA
Caption: Fig. 2. Standard curve with the correlation coefficient ([r.sup.2]) ob- tained by plotting the cycle threshold (Ct) against the input DNA plasmid quantity (logarithm scale) after real-time PCR
Table 1. Trichoderma isolates used in this study S. Strain Source ITCC No. Fungus No. code Identified 1 6 CP Sultanpur 7442/09 T.atroviride 2 24CP Sitapur 7443/09 T.atroviride 3 71 L Hardoi 7445/09 T.atroviride 4 115 L Bahraich 7446/09 T.atroviride 5 52 L Unnao 7447/09 T.atroviride 6 105 CP Etawah 7451/09 T.atroviride 7 75 PP Auriya 7448/09 T.atroviride 8 126 PP Kanpur Dehat 7449/09 T.atroviride 9 21 PP Kaushambi 7437/09 T. longibrachiatum 10 31PP Allahabad 7438/09 T. longibrachiatum 11 81PP Mirzapur 7439/09 T. longibrachiatum 12 100 PP Sonbhadra 7440/09 T. longibrachiatum 13 120 PP Bhadoi 7441/09 T. longibrachiatum 14 28 CP Barabanki 7444/09 T. longibrachiatum 15 5 CP Kanpur Nagar 7450/09 T. longibrachiatum 16 8 CP Kanpur Nagar 8305/11 T. viride 17 11 CP Allahabad 8306/11 T. viride 18 17 CP Kaushambi 8307/11 T. viride 19 33 CP Sitapur 8308/11 T. viride 20 34 CP Hardoi 8309/11 T. viride 21 35 CP Fatehpur 8310/11 T. viride 22 66 CP Auriya 8311/11 T. viride 23 89 CP Unnao 8312/11 T. viride 24 102 CP Etawah 8313/11 T. viride 25 119 CP Kanpur Dehat 8314/11 T. viride 26 01 PP Hardoi 8315/11 T. viride 27 09 PP Kanpur Nagar 8316/11 T. viride 28 14 PP Barabanki 8317/11 T. viride 29 17 PP Fatehpur 8318/11 T. viride 30 29 PP Unnao 8319/11 T. viride 31 42 PP Bahraich 8320/11 T. viride 32 67 PP Auriya 8321/11 T. viride 33 74 PP Sitapur 8322/11 T. viride 34 78 PP Etawah 8323/11 T. viride 35 124 PP Kanpur Dehat 8324/11 T. viride 36 13 L Kanpur Nagar 8325/11 T. viride 37 104 L Etawah 8331/11 T. viride 38 35 L Sitapur 8326/11 T. viride 39 68 L Hardoi 8327/11 T. viride 40 74 L Auriya 8328/11 T. viride 41 89 L Gonda 8329/11 T. viride 42 100 L Faizabad 8330/11 T. viride 43 109 L Allahabad 8332/11 T. viride 44 117 L Sultanpur 8333/11 T. viride 45 119 L Kanpur Dehat 8334/11 T. viride 46 Th azad CSA Univ. 6796/12 T.harzianum Farm 47 T.spe CSA Univ. 8940/12 T.asperellum /(CSAU) Farm 48 T. kon CSA Univ. 5201/13 T.koningii /(CSAU) Farm 49 T. vire CSA Univ. 4177/13 T. virens /(CSAU) Farm S. Crop GPS Location No. 1 Chickpea Latitude: 26.2500 [degrees]N Longitude: 79.0000 [degrees]E 2 Chickpea Latitude: 27.5700 [degrees]N Longitude: 80.6800 [degrees]E 3 Lentil Latitude: 26U 292 28.3232 2 Longitude: 80U 182 26.3612 2 4 Lentil Latitude: 27.7500 [degrees]N Longitude: 81.7500 [degrees]E 5 Lentil Latitude: 26.5500 [degrees]N Longitude: 80.4800 [degrees]E 6 Chickpea Latitude: 26.7700 [degrees]N Longitude: 79.0300 [degrees]E 7 Pigeon pea Latitude: 26.4700 [degrees]N Longitude: 79.5200 [degrees]E 8 Pigeon pea Latitude: 26.2277 Longitude: 79.8370 9 Pigeon pea Latitude: 26U 342 27.612 2 Longitude: 79U 182 24.6232 2 10 Pigeon pea Latitude: 25.4358 Longitude: 81.8463 11 Pigeon pea Latitude: 25.1500 [degrees]N Longitude: 82.6000 [degrees]E 12 Pigeon pea Latitude: 24.6897 [degrees]N Longitude: 83.0653 [degrees]E 13 Pigeon pea Latitude: 25.3932 Longitude: 82.5657 14 Chickpea Latitude: 26.9200 [degrees]N Longitude: 81.2000 [degrees]E 15 Chickpea Latitude: 25U 82 34.8212 2 Longitude: 81U 592 2.9792 2 16 Chickpea Latitude: 26.4600 [degrees]N Longitude: 80.3300 [degrees]E 17 Chickpea Latitude: 25.4358 Longitude: 81.8463 18 Chickpea Latitude: 26U 342 27.612 2 Longitude: 79U 182 24.6232 2 19 Chickpea Latitude: 27.5700 [degrees]N Longitude: 80.6800 [degrees]E 20 Chickpea Latitude: 26U 292 28.3232 2 Longitude: 80U 182 26.3612 2 21 Chickpea Latitude: 25.9300 [degrees]N Longitude: 80.8000 [degrees]E 22 Chickpea Latitude: 26.4700 [degrees]N Longitude: 79.5200 [degrees]E 23 Chickpea Latitude: 26.5500 [degrees]N Longitude: 80.4800 [degrees]E 24 Chickpea Latitude: 26.7700 [degrees]N Longitude: 79.0300 [degrees]E 25 Chickpea Latitude: 26.2277 Longitude: 79.8370 26 Pigeonpea Latitude: 26U 292 28.3232 2 Longitude: 80U 182 26.3612 2 27 Pigeonpea Latitude: 25U 82 34.8212 2 Longitude: 81U 592 2.9792 2 28 Pigeonpea Latitude: 26.9200 [degrees]N Longitude: 81.2000 [degrees]E 29 Pigeonpea Latitude: 25.9300 [degrees]N Longitude: 80.8000 [degrees]E 30 Pigeonpea Latitude: 26.5500 [degrees]N Longitude: 80.4800 [degrees]E 31 Pigeonpea Latitude: 27.7500 [degrees]N Longitude: 81.7500 [degrees]E 32 Pigeonpea Latitude: 26.4700 [degrees]N Longitude: 79.5200 [degrees]E 33 Pigeonpea Latitude: 27.5700 [degrees]N Longitude: 80.6800 [degrees]E 34 Pigeonpea Latitude: 26.7700 [degrees]N Longitude: 79.0300 [degrees]E 35 Pigeonpea Latitude: 26.2277 Longitude: 79.8370 36 Lentil Latitude: 26.4600 [degrees]N Longitude: 80.3300 [degrees]E 37 Lentil Latitude: 26.7700 [degrees]N Longitude: 79.0300 [degrees]E 38 Lentil Latitude: 27.5700 [degrees]N Longitude: 80.6800 [degrees]E 39 Lentil Latitude: 26U 292 28.3232 2 Longitude: 80U 182 26.3612 2 40 Lentil Latitude: 26.4700 [degrees]N Longitude: 79.5200 [degrees]E 41 Lentil Latitude: 27.2500 [degrees]N Longitude: 82.0000 [degrees]E 42 Lentil Latitude: 26.7800 [degrees]N Longitude: 82.1300 [degrees]E 43 Lentil Latitude: 25.4358 Longitude: 81.8463 44 Lentil Latitude: 26.2500 [degrees]N Longitude: 79.0000 [degrees]E 45 Lentil Latitude: 26.2277 Longitude: 79.8370 46 Chickpea Latitude: 26.4912 [degrees]N Longitude: 80.3070 [degrees]E 47 Pigeonpea Latitude: 26.4912 [degrees]N Longitude: 80.3070 [degrees]E 48 Pigeonpea Latitude: 26.4912 [degrees]N Longitude: 80.3070 [degrees]E 49 Pigeonpea Latitude: 26.4912 [degrees]N Longitude: 80.3070 [degrees]E
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|Author:||Shahid, Mohammad; Srivastava, Mukesh; Pandey, Sonika; Kumar, Vipul; Singh, Anuradha; Trivedi, Shubha|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Mar 1, 2016|
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