Research Article
Symbiotic N Fixation: Nodulation of Tepary Bean (Phaseolus acutifolius A. Gray)1
Chinna D Mapp1, Regina Knight-Mason2 and Harbans L Bhardwaj 3,4*
1Contribution of Virginia State University, Agricultural Research Station. The use of trade names or vendors does not imply approval to the exclusion of other products or vendors that may also be suitable
2Former MS (Biology) degree student, Department of Biology, Virginia State University, Petersburg, VA 23806 USA
3,4Department of Biology, Virginia State University, Petersburg, VA 23806 USA
Corresponding author: Harbans L Bhardwaj, Agricultural Research Station, Virginia State University, PO Box9061, Petersburg, VA 23806, USA; E-mail: HBHARDWAJ@VSU.EDU
Citation: Mapp CD, Knight-Mason R, Bhardwaj HL. Symbiotic N Fixation: Nodulation of Tepary Bean (Phaseolus acutifolius A. Gray). J Plant Sci Res.2016;3(1): 151.
Copyright © Bhardwaj HL 2016 et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Journal of Plant Science & Research | ISSN: 2349-2805 | Volume: 3, Issue: 1
Submission: 08/06/2016; Accepted: 24/06/2016; Published: 01/07/2016
Abstract
Tepary Bean (Phaseolus acutifolius A. Gray), is known to be a drought-tolerant grain legume and could play a positive role in supporting sustainable role in production of food and feed under changing climatic conditions due to its’ ability to Symbiotically fix atmospheric N. However, existence of any rhizobial strain that could nodulate Tepary bean was unknown before the initiation of this study. We evaluated nodulation in 51 Tepary bean accessions after inoculation with a rhizobial strain (Phaseolus Spec #3) from Novozymes BioAg, Inc. to determine existence, if present, of nodulation. Our results indicated that Tepary bean could be successfully nodulated by using Phaseolus Spec #3 strain. We grew 51 Tepary bean accessions in a mixture of sand and vermiculite in two experiments and inoculated seeds with Phaseolus Spec #3 and recorded data on nodule size scores (1= small sized nodules similar in size to mustard seed; 2= medium sized nodules; varying from mustard seed size to soybean seed size and 3= large sized nodules greater in size than soybean seed), nodule number scores (1= less than 5 nodules; 2= more than 10 nodules; and 3= more than 20 nodules). A SPAD (SPAD-502, Minolta Corporation) meter was used to record relative chlorophyll in each plant prior to harvesting. A new variable named “Combined Score” was computed by summing values for nodule sizescores, nodule number scores, and SPAD readings. The SPAD unit reading, nodule size score, nodule number score, and combined score means were27.08, 1.48, 2.12, and 30.68, respectively. SPAD reading exhibited 0.44 and 0.61 correlation coefficient (significant at 1% level), respectively with nodule size score and module number score. Based on results of this study, we conclude that Tepary bean can be nodulated by Phaseolus Spec #3 rhizobial strain.
Keywords: Symbiotic N Fixation; Bradyrhizobium; Phaseolus acutifolius; Nodule number; Nodule size; SPAD reading
Introduction
Tepary bean (Phaseolus acutifolius A. Gray) is a desert legumenative to the south western United States, Texas, New Mexico andMexico. Tepary bean has served as a staple food for generations ofprehistoric Native Americans [1]. Tepary bean’s dry seed is consumeddue its rich protein and carbohydrate content. “Tepary bean’s dry seedis consumed due to its rich protein content varying from 17 to 27percent and due to its rich carbohydrate content averaging 59 percent”[2]. Biochemical analysis of Tepary bean indicates that Tepary plantsproduce a high amount of soluble solids in the seed such as glucose and sucrose under sufficient or insufficient water supply [3]. Teparybean plants flower within 27- 40 days after germination and ripenbetween 60-80 days [4]. Short duration wild Tepary bean varietiesmature within two months in tropical climates. However, its growthperiod may extend up to 120 days in cooler areas such as in costalAlgeria [4].
The United Nations Food and Agriculture Organization estimatesthat about 795 million people of the 7.3 billion people in the world,or one in nine, were suffering from chronic undernourishment in2014-2016 (http://www.worldhunger.org/articles/Learn/world%20 hunger%20facts%202002.htm). In many parts of the world there isincreasing scarcity of water for agriculture [5]. Consequently, thereis potential for the use of drought-tolerant legumes in agriculture notonly for their Symbiotic N Fixation potential but also for reducedwater use.
Tepary bean is considered one of the most heat and droughttolerant crops in the world [2]. This legume has the ability to flourishduring intense drought with low annual rainwater requirementsranging from 500 mm to 1700 mm [4]. Tepary bean has beenintroduced to African agriculture due to its ability to mature from asingle thunderstorm’s down pour or single irrigation [4]. Researchersin Virginia have demonstrated that Tepary bean has the ability toproduce well in the southern USA [6].
If proven successful, Tepary bean can be used as a sustainablecrop for environmentally-friendly agriculture by lowering grower’sdependence on synthetic nitrogen fertilizers. The use of nitrogen fixedby the Tepary bean may eliminate and or reduce applying syntheticinorganic nitrogen fertilizer, thus reducing the possibility of waterpollution caused by run-off into streams, lakes, rivers and tributaries.
This study was conducted during 2006 and 2007 as a MS degreethesis project to identify rhizobial strains that can nodulate Teparybean. At this time, no rhizobial strain was known to nodulate Teparybean.
Materials and Methods
Before initiation of this study, existence of any rhizobial strainthat could nodulate Tepary bean was unknown. After severalattempts, EMD Crop BioScience, Inc. (Now Novozymes BioAg,Inc., Milwaukee, Wisconsin USA 53209) identified one rhizobialstrain (Phaseolus Spec #3) as a potential inoculent for tepary beanand provided this strain for our studies. This strain was a mixture ofNitragin strain numbers 127D3, 127E12, and 127N2. Nodulation in51 accessions of Tepary bean was studied in two experiments during2006 and 2007 using Phaseolus Spec #3 rhizobial strain. The firstexperiment was planted on February 1, 2006 and harvested on March11, 2006. The second experiment was planted on November 26, 2007and harvested on January 22, 2008.
These experiments were conducted in the greenhouse at theRandolph Agricultural Research Station, Petersburg, Virginia. The experimental design used was a completely random design with threereplications. Each combination of tepary line and bradyrhizobialstrain Phaseolus Spec #3 was planted in three different pots (threereplications). Thirty cm tall plastic pots were filled with moistenedplanting material (1:1 sterilized vermiculite and filtered sand). Twoseeds were placed in each pot in a hole about 3 cm deep. A pipettewas used to draw the liquid inoculant mixture out of a sterilecontainer and then released from the pipette on the seed. This wasdone repeatedly for every set of three of the 51 lines. After saturation,the seeds were immediately covered with planting material and thenwatered liberally.
Data recording
At harvest, plants were harvested, their roots gently washed, andnodule size and nodule number scores were recorded each using thefollowing scale: Nodule size scores (1= small sized nodules similar insize to mustard seed; 2= medium sized nodules; varying in size frommustard to soybean seed and 3= large sized nodules greater in size than soybean seed), nodule number scores (1= less than 5 nodules;2= more than 10 nodules; and 3= more than 20 nodules). A SPAD(SPAD-502, Minolta Corporation) meter was used to record relativechlorophyll in each plant prior to harvesting. A new variable named“Combined Score” was computed by summing values for nodule sizescores, nodule number scores, and SPAD readings.
All data were analyzed using SAS [7].
Results and Discussion
Results indicated that significant variation existed among 51 tepary bean accessions, after inoculation with rhizobial strain PhaseolusSpec #3, for all traits under consideration (Table 1) except for nodulesize score, which was only significant at 10 % level of significance.The SPAD unit reading, nodule size score, nodule number score, andcombined score means were 27.08, 1.48, 2.12, and 30.68, respectively.Values of Coefficients of Determination varied from 70 to 91 percentindicating that variation among 51 Tepary bean accessions accounted for major variation in traits under consideration.
Table 1: Symbiotic N fixation related traits of tepary bean accessions upon inoculation with Phaseolus Spec #3 rhizobial strain.
We compared morphological traits of Tepary bean accessions(Table 2) and observed that plant growth type (erect or viny) did notaffect nodulation traits in Tapery bean whereas existence of narrowleaves imparted significant advantage for nodulation traits over broadleaves: narrow leaved-accessions had significantly higher means for SPAD unit readings than accessions for broad leaves (28.70 vs. 26.23,respectively), nodule size score (1.57 vs. 1.43, respectively), andcombined score (32.40 vs. 29.78, respectively). Accessions with blackseeds had significantly higher SPAD unit readings and combinedscores.
Table 2: Symbiotic N fixation traits of 51 tepary bean accessions upon inoculation with Phaseolus Spec #3 rhizobial strain.
Country of origin of Tepary bean accessions (Table 3), affected nodulastion traits: accessions originating from Puerto Rico had 38.8SPAD unit reading and 42.69 combined score both being significantlyhigher than for accessions originating from Mexico, Costa Rica, USA,Nicaragua, or El Salvador. Tepary bean accessions originating fromthese countries had SPAD unit readings and combined scores of26.94 and30.49, 26,22 and 29.88, 25.96 and 29.53, 24.60 and 28.80,and 21.38 and 24.98, respectively.
The combined score also showed that of the 51 tepary beanaccessions in the study, PI-535226 was the best cultivar for traitsrelated to nodulation and nitrogen fixation. Using combined score,developed by combining various traits, the best tepary bean lines werePI-535526, PI-440789, PI-535227, PI-533454 and PI-440804. Basedon the combined score, PI-535226 was most efficient for nodulationcharacteristics with a combined score of 50.98, followed by PI-440789with a combined score of 42.44.
The Minolta Chlorophyll Meter (SPAD-502) has been extensivelyused to instantly measure the amount of chlorophyll content; anindirect measure of nitrogen content (http://www.specmeters.com/Chlorophyll_Meters/Minolta_SPAD_502_Meter.html). In thesestudies, mean SPAD unit readings varied from 17.0 to 47.3 with amean of 27.08 (Table 1). The ten lines with highest SPAD unit readingswere PI-535226, PI-440789, PI-535227, PI-533454, PI-440804, PI-440806, PI-440802, PI-533453, PI-440788, and PI-440803. Given thatSPAD unit readings are related to nitrogen content of the leaves, wecan conclude that these five lines benefited most from inoculationwith the Phaseolus Spec #3 inoculum.
The means presented in Table 1 indicate existence of significantvariation among 51 tepary bean accessions. This observation alongwith the observation that three accessions were observed to haveSPAD unit readings beyond the positive side of mean ± 1 standarddeviation demonstrate that some tepary bean accessions hadfunctioning roots, otherwise these accessions could not have leavesgreener than other accessions given that the only source of nitrogenwas that from nodules.
Significant correlation existed between SPAD readings, nodulenumber score, and nodule size scores. SPAD readings exhibited 0.44 and 0.61 Pearson correlation coefficients (significant at 1% level),respectively with nodule size and nodule number score. Apparently,accessions with higher root nodules size or root nodule numberimplied functionality of these nodules by increased SPAD unitreadings.
Based on results of this study, we conclude that Tepary can benodulated by Phaseolus Spec #3 rhizobial strain. We spoeculate thatit is possible that other rhizobial strain might be able to nodulateTepary bean and this research should be expanded. It is also possiblethat identification of additional rhizobial strains for nodulation inTepary bean could lead to existence of specificity between rhizobialstrains and Tepary bean lines similar to what we observed in whitelupin (Lupinus albus L.) during our previous research period [8]. Ourresults are contrary to those of Duggar.
Acknowledgement
We are thankful to Mr. John Kosanke (Novozymes BioAg, Inc.,Milwaukee, Wisconsin, USA 53209), for provideing the PhaseolusSpec #3 rhizobial strain. Financial assistance to Chinna Mapp byVirginia State University is also thankfully acknowledged.
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