Short communication
Use of a standard TWAIN scanner and software for nodule
number determination on dierent legume species
M. de A. Lira Jr.
a, D.L. Smith
b,*
a
Departamento de Agronomia, Universidade Federal Rural de Pernambuco, Brazil
b
Plant Science Department, McGill University, Macdonald Campus, 21111 Lakeshore Road, Ste. Anne de Bellevue, Que., Canada H9X 3V9
Received 29 July 1999; received in revised form 14 February 2000; accepted 23 February 2000
Abstract
Biological nitrogen ®xation by legume±Rhizobium system is the main natural source of nitrogen in agricultural ®elds. To measure nodule number, pea (Pisum sativum), bean (Phaseolus vulgaris) and lentil (Lens culinaris) were planted in plastic pouches. These pouches allowed root system observation. For each plant, roots were scanned, nodules hand separated, scanned and hand counted. All scanner methods of counting had correlation coecients with hand counting above 0.85, and were highly signi®cant.72000 Elsevier Science Ltd. All rights reserved.
Keywords: Rhizobium; Nodule; Method; Image analysis; Development
1. Introduction
Biological nitrogen ®xation by the legume±Rhizo-bium system is the main natural source of nitrogen in agricultural systems. Number of nodules is one of the most important parameters of this symbiosis. Nodule population dynamics is very important, but has been dicult to measure due to its destructive and labor intensive nature, coupled with the nodules' quick decomposition.
One unexplored possibility is using standard compu-ter equipment to scan the roots, allowing lacompu-ter measurement of nodules. This was attempted for bean using proprietary hardware and software by Vikman and Vessey (1993).
2. Materials and methods
Seeds of bean, lentil and pea were surface sterilized according to Montealegre et al. (1995), and trans-planted at day 5 after germination into plastic pouches (cyg germination pouches Ð MegaInternational, Min-neapolis, MN). These were held in a water bath, with water temperature controlled at 208C, and air tempera-ture at 258C/168C (16/8 h day/night cycle). The pouches were ®lled with standard Hoagland solution (Hoagland and Arnon, 1950); this was replaced weekly.
Two days after transplanting, bean plants were inoculated with Rhizobium leguminosarum bv. phaseoli strain 127K105, and pea and lentil received R.
legumi-nosarum bv. viceae strain 175G106 (both from
Lipha-tech, Milwaukee, WI, USA). Inoculation was done with 1 ml of a saline solution (0.85% NaCl in distilled water) per seedling with bacterial culture suspended to an optical density of 0.1 at 640 nm, following Pan et al. (1998).
Soil Biology & Biochemistry 32 (2000) 1463±1467
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Samples were taken at days 27, 33 and 40 after in-oculation. Each was scanned with a Plustek OpticPro 4800P71or an Umax Astra 610S7scanner, at 24-bit color and 100 dots per inch (dpi) resolution.
Before scanning, pouches were emptied of solution, and the scanner's cover was closed. There was no cor-rection due to possible dierential height from the
glass plate, the assembly being considered solely two-dimensional.
The ®rst two scans (Pouch) were done on the front (Fig. 1a) and back of the pouch. For the sec-ond method (Paper), the scan was made after the paper ¯ap (Fig. 1a, arrow) was removed. The third method (Root) was executed on roots after removal from the pouch. For the fourth method (Separated), the scan was conducted on nodules removed manu-ally. Separated nodules were also hand counted (Hand).
Each nodule was ``painted'' in black (Fig. 1b), using a Wacom PenPartner7 tablet with Corel PhotoPaint
Fig. 2. Scatter plots of nodule number determinations by hand or by dierent scanning procedures (Pouch Ð nodules counted in the pouch scan, Paper Ð nodules counted in the pouch scan with the paper strip removed, Root Ð nodules counted on the isolated roots scan, Separated Ð nodules counted in the scan of hand separated nodules).
1
The products are copyright of the owner; and use of a commer-cial name indicates the product actually used during this experiment, and does not represent indication of exclusive adequacy of the pro-duct for this application.
77, and a threshold was applied so the ®le showed only the nodules (Fig. 1c).
These ®les were used on Scion Image7with macros for automatic count of nodules (Fig. 1d). The results formed a text ®le for each scan.
These ®les were the input to SAS7 (SAS Institute, 1990), used to summarize in text ®les with number of nodules and descriptive statistics for each plant.
Quattro Pro 87 was used to consolidate data and resubmit them to SAS for correlation and regression analysis. The hand-counted values were the dependent variables and results from the scanned ®les were the independent variables, one method/hand pair at a time.
3. Results
All procedures resulted in highly signi®cant corre-lations between scanner estimated nodule number and hand-counted nodule number. These correlations var-ied between 0.85 (Paper) and 0.98 (Separate) (Fig. 2).
The scatter plots generated good concentrations of the points, especially for Separated method (Fig. 2). For Pouch and Paper methods, the slopes were stee-per, as indicated by the linear equations.
4. Discussion
The only reference using image analysis for legume nodule analysis (Vikman and Vessey, 1993) used a pro-prietary system which divided the nodules into four categories, aiming at determining which nodule class contributed most to nitrogen ®xation. As such it was more limited in its approach. Vikman and Vessey (1993) measured an R2of 0.88 for hand and machine classi®ed nodules, which is comparable to the relation-ships obtained in our work.
The lowerR2values for Root, Paper and Pouch can be explained by some nodules being obscured by roots, which did not happen for Separated nodules. This is corroborated by higher regression coecients for both Paper and Pouch methods indicating some nodules were not seen or identi®ed. The highest regression coecient for Pouch corroborates observations that some nodules were behind the paper ¯ap.
It is important that users are trained and adopt a visual standard for a nodule. This is especially import-ant when the nodules are attached to roots, when visual confusion may occur more easily. Even for trained users, it would be advisable to organize them in such a way that any dierences can be detected using standard statistical techniques.
Although all methods had high R2 values, the best method for nodule counting was hand separation. This method is destructive, thus removing some of the pre-cision from repeated measures and increasing the num-ber of plants in experiments where nodule population development is examined over time. This also indicates that the Separated method is highly applicable in other experiments, such as ®eld grown plants.
The high R2s observed for the Paper method indi-cate that it can be used as a non-destructive technique. This allows the possibility of following one cohort of nodules from formation until senescence, if the limi-tations of growth without a solid media can be accepted as reasonable for the objectives of the study.
This speci®c system was chosen based on its wide availability and low cost, since Scion Image7 is free-ware and the remaining components have multiple uses, and can be readily replace by any other item in current use. This choice incurs in some drawbacks lim-iting some features, such as the use of graphical layers on the painting program, if so desired.
A commercially available program, such as SigmaS-can Pro7, would reduce the number of necessary steps, with the painting being done on separate layers inside SigmaScan Pro7. If this use is viable, it should somewhat simplify the procedure, but the combination of freely available and generally used programs proved to be feasible.
Finally, the technique we describe allows for detailed nodule measurements with the same eort necessary for nodule counting. If the nodules are separated from the root to obtain dry weight, scanning will make it possible to have a permanent record of their appear-ance and size. Although direct scanning of roots in pouches resulted in a lower R2 than observed for manually separated nodules, the results were accepta-bly accurate and allow non-destructive nodule count-ing and measurcount-ing.
Acknowledgements
This experiment was supported in part by CNPq -Brazil, Grant #200536/95-8 and in part by a Natural Sciences and Engineering Research Council of Canada grant held by D. Smith. We wish to thank the col-leagues from the Crop Physiology Laboratory, Plant Science Department, McGill University for their sup-port.
References
Montealegre, C., Graham, P.H., Kipe-Nolt, J.A., 1995. Preference in the nodulation of Phaseolus vulgaris cultivar RAB39. Canadian Journal of Microbiology 41, 992±998.
Pan, B., Zhang, F., Smith, D.L., 1998. Genistein addition to the rooting medium of soybean at the onset nitrogen ®xation increases nodulation. Journal of Plant Nutrition 211, 1631±1639.
SAS Institute, 1990. SAS Language. Reference Version 6, 1042 pp. Vikman, P.A., Vessey, J.K., 1993. Ontogenetic changes in root
nodule subpopulations of common bean (Phaseolus vulgarisL.). Part III: Nodule formation, growth and degradation. Journal of Experimental Botany 44, 579±586.
