Y Moutia, M Mangar, M Teeluck, S Sakurdeep, LJC Autrey and S Saumtally Mauritius Sugar Industry Research Institute
ABSTRACT
The acquisition of a video image analyser (VIA) paved the way to the development of numerous applications in the field of plant pathology as well as in other fields. The equipment was used to develop a reliable method for evaluating the percentage leaf infection by the sugar cane common rust pathogen (Puccinia melanocephala). This had previously been performed by visual examination with a degree of human subjectivity. In the same field, the measurement of uredospores of the rust pathogen allowed its spore size distribution to be rapidly determined with greater accuracy than with the use of a micrometer scale. In plant breeding, pollen grains were measured to test whether size was related to fertility. In irrigation, losses due to wind drift under a centre pivot irrigation system were estimated by measuring the size of droplets collected on water sensitive paper. In sugarcane technology, the measurement of sugar crystals at monthly intervals was carried out for several sugar estates to verify whether their sieves were performing well or required maintenance. For all these applications, the use of the VIA not only proved to be fast and reliable but also more objective. Keywords: leaf infection, spore size, common rust, pollen grains, irrigation droplets, sugar crystals, video image analysis
In this paper, the use of VIA is compared in terms of accuracy and rapidity to conventional methods for the following applications: quantification of sugar cane leaf area infected by common rust, spore size distribution of the rust pathogen, wind drift losses of sprinklers, pollen fertility assessment and measurement of sugar crystals.
The crystal size distribution of the C-massecuite (a brown viscous material in which sugar crystals are embedded) is therefore of prime importance to the final molasses exhaustion performance. The shape
AMAS 2001. Food and Agricultural Research Council, Rduit, Mauritius. 10
and the size of a crystal have a direct influence on molasses removal and the effectiveness of the washing operation in the continuous centrifugals.
The software incorporates several subroutines that can be chosen to build a program that will then perform each task in an automated way. Figure 1 Components of the video image analysis system
Screening of seedlings
In order to develop the methodology for screening seedlings against rust, progenies from two biparental crosses were inoculated (Moutia 1994, Autrey et al. 1996) and leaf samples assessed after four weeks. The parents used in the crosses were: 1. 2. M 555/60 (highly susceptible) x M 1030/71 (highly susceptible) M 695/69 (slightly susceptible) x M 1030/71 (highly susceptible)
Pollen fertility
Pollen grains of eight commercial male varieties (Table 1) were released from anthers (prior to dehiscence) in a drop of Alexanders stain mounted on a slide and observed at a magnification of x50. Pollen size and Aspect Ratios were determined. Three replicates of 25 pollen per variety were measured for length, breadth and ferret diameter (average of 36 calliper measurements at intervals of 5). The dimensionless expression called Aspect Ratio was derived by dividing the length of each pollen by its breadth. A value of 1 would represent a perfect circle whereas lower and higher values would indicate an oval shape.
Table 1 Relationship between pollen diameter, aspect ratio and pollen fertility for eight male varieties Diameter Mean SE m 51.6 1.5 50.2 0.5 37.6 2.8 45.1 4.5 43.7 1.7 43.5 2.3 39.4 1.2 36.8 0.2 Aspect Ratio(L:B) Mean SE 1.079 0.008 1.087 0.007 1.008 0.006 1.121 0.025 1.170 0.080 1.108 0.024 1.101 0.017 1.099 0.072 Pollen fertility % 39.7 18.6 38.0 25.0 13.4 19.1 6.6 0
Person 1
Person 2
VIA
probability of making the right categorisation/decision given the disease percentage observed on the indicator leaf or leaves. Results (Table 3, Figure 3) revealed that only leaf number 3 and some leaf combinations (leaves 1 and 4 or leaves 2 and 4) were good estimators of plant mean infection. Table 2 Comparison of simulated percentages to the estimations of two persons and that of the VIA
Estimated Percentage True % 0 1 2 4 5 7 10 12 15 20 23 25 28 30 33 35 38 45 48 50 52 55 60 63 65 70 75 78 82 100 Person 1 Person 2 VIA st nd st nd st nd 1 estimation 2 estimation 1 estimation 2 estimation 1 estimation 2 estimation 0 0 0 0 0 0.4 1 1 1 1 2.3 1.9 1 1 1 2 3.4 3.6 2 8 5 2 5.5 6.7 5 5 10 5 7.9 7.3 5 5 10 5 11.8 10.4 10 5 10 10 13.4 13.5 10 10 10 10 16.9 16.1 20 10 20 20 19.0 18.8 15 15 25 20 24.4 24.5 20 15 30 20 27.9 27.2 15 15 30 20 29.1 30.0 25 25 30 20 33.1 32.4 25 20 25 20 33.7 34.5 25 25 30 30 35.6 35.2 45 30 30 30 39.2 38.0 35 40 40 30 42.3 43.0 60 45 50 30 49.2 50.1 30 35 50 40 48.9 48.3 65 45 50 40 51.2 51.0 50 55 50 40 52.0 51.8 35 50 50 40 55.1 54.0 70 40 60 60 58.3 58.1 70 60 60 60 62.2 62.0 75 65 60 70 64.5 63.7 80 75 60 60 68.2 68.5 75 70 70 80 70.8 70.6 80 80 80 80 78.7 77.5 80 85 80 90 77.3 76.5 100 100 100 100 99.8 99.6
Table 3 Probabilities of making the right classification / decision and of making error type (1) based on one leaf or a combination of leaves as representative of the plant Leaf / Leaf combination Leaf number 3 Mean of leaves 1 & 3 Mean of leaves 1 &4 Mean of leaves 2 & 4 Mean of leaves 1,2 & 4 Mean of leaves 2,3 & 4 Mean of leaves 1,2,3 & 4 Probability of making the right classification / decision 0.87 0.82 0.90 0.90 0.90 0.87 0.94 Probability of making error type (1) 0.01 0.05 0.01 0.01 0.03 0.02 0.02
Pollen fertility
Mean diameter was variable between varieties ranging from 36.8 + 0.2 m in variety M 1182/77 to 51.6 + 1.5 m in variety M 245/76. Aspect ratio was, however, less variable ranging from 1.008 + 0.006 m to 1.170 + 0.080 m in variety SP 71799. Pollen fertility varied from 0% in variety M 1182/77 to 39.7% in variety M 245/76 (Table 1).
60
50
40 % Frequency
30
20
10
0 15-20 20-25 25-30 30-35 35-40 Range ( m ) Breadth 40-45 45-50 50-55
Length
Table 4 Length and breadth of droplet stains (m) measured with VIA compared to measurements made with a graticule Measurements with VIA Length Breadth 2 151.0 1 851.0 860.7 716.8 920.1 719.4 1 302.0 986.1 776.2 587.4 1 205.0 1 073.0 1 472.0 961.0 2 123.0 990.0 1 483.0 1 257.0 1 271.0 1 125.0 Measurements with graticule Length Breadth 2 100 1 700 1 000 900 900 700 1 200 1 000 700 600 1 100 1 100 1 400 900 2 000 800 1 500 1 300 1 200 1 100
Table 5 Wind drift losses from a centre pivot for five wind speeds Wind speed -1 ms 1.38 2.22 5.27 6.11 6.94 Drift losses % at 20 m at 25 m 0.21 0.11 0.20 0.10 0.91 0.61 2.15 0.97 2.89 1.79
For C-massecuite samples from different factories (Table 7), the EQ ranged from 0.169 - 0.304 mm. The maximum coefficient of variation obtained was 29% which is reasonable for such measurements. Table 7 Measured parameters for C-massecuite samples from 11 sugar factories Mean equivalent diameter mm 0.263 0.169 0.238 0.252 0.274 0.304 0.242 0.269 0.252 0.173 0.201
Factory 1 2 3 4 5 6 7 8 9 10 11
Mean breadth mm 0.280 0.166 0.225 0.257 0.289 0.304 0.251 0.272 0.233 0.174 0.190
CV % 13 26 26 22 25 29 17 18 22 16 21
which stipulates that people recognize a change in a certain value only if the ratio between the change and the initial value is greater than a minimum constant (Ahituv and Neumann 1989).
Although both are undesirable, the consequence of error (1) is far reaching, involving the possibility of releasing a susceptible variety for commercial planting with the risk of significant losses at field level. Therefore, in the present model it was important to identify the possible indicator leaf/leaves that gave the least probability of error (1) whilst at the same time ensuring good classification and practicality. Leaf number 3 was the most promising candidate with 87% right classification and 1% of error (1). It was also clear from these results that increasing the number of leaves in the sample did not improve the accuracy of classification to a great extent. In practice, measurement by VIA at the seedling stage would have to be supplemented by field observations of the same clones, planted from cuttings, in order to eliminate susceptible ones that could have escaped the initial screening.
Pollen fertility
The strong positive correlation (r2=0.72) between diameter and fertility indicates that this parameter can be a useful one to categorise male varieties into fertility classes. In Cuba, Guerra et al. (1989) noted that highest fertility was observed in commercial varieties with pollen diameters in the range of 30-40 m. Although a negative correlation was noted between Aspect Ratio and pollen fertility, it was too low (r2=0.12) to be significant. A larger number of varieties with a wider range of Aspect Ratios would have to be analysed in the future to confirm/infirm the hypothesis that pollen fertility is related
to aspect ratio. It would also be desirable to test whether the percentage of each pollen stained has an effect on viability and hence on fertility. This could be adequately tackled by VIA.
CONCLUSION
The VIA is a very versatile equipment which can perform quickly an array of tasks. The program included with such systems has many sub-routines. These include many mathematical transformations which enhance the raw image to facilitate processing. However, these transformations should be used only if necessary as they can sometimes affect results in a negative way. It is of utmost importance to stress that the performance of the system relies a lot on the ability of the user to prepare very good images especially as regards to uniform illumination. The applications developed at the MSIRI are only a small fraction of what the system can do. From the results obtained, it is evident that the use of VIA improved considerably on accuracy and rapidity in all the fields tested. The VIA could also be used in the future for the assessment of other foliar sugar cane diseases as well as in the preparation of field assessment keys in cases where they are not available such as for brown spot (Cercospora longipes) and yellow spot (Mycovellosiella koepkei). Other future applications include preparing karyotypes, scanning gels, automatic selection of interesting cell cultures in monoclonal antibody production and root surface area analysis.
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