Spectral properties of strawberry plants

Strawberry (Fragaria x ananassa Duch.) is very important small fruit in Hungary. Strawberry is one of the earliest fresh fruits after the wintertime poor fruit supply in the market, and a delicious fruit not only for finished products, but also for ingredients to be included in complex foods such as ice-creams, cereals, dairy, confectionery, and bakery products (Dermesonlouoglou, 2006). The deep frozen products, jam, and preserved fruit are also popular. Breeders have been producing several strawberry varieties with a broad range of capabilities. The most important qualities are the good level of resistance to Phytophtora cactorum, Ph. fragariae, Botrytis cinerae, and other strawberry diseases, high fruit quality, regular shape, long shelf-life, good smell and flavour, and attractive colour (Roudeillac, 1993). The different varieties, however, react differently to environmental and growing conditions. Traditionally the evaluation of the yield of the plant is based on measurements of the fruit size and quantity and on organoleptic evaluation of the fruit quality (Nestby, 1998, Miner et al., 1997, Darbellay et al., 2000). However, this is a tedious process, moreover sensory evaluation always has a certain amount of subjectivity. In an extensive statistical evaluation Jacquemoud et al., (1995) investigated the correlation of the biochemical properties of 73 plant leaves of different species with their optical properties. They found good prediction performances for the protein, cellulose, and lignin content. Based on these correlations, radiative transfer models (Jacquemoud et al., 1996) were constructed which make it possible to calculate the simulated spectral response of a leaf model. Through the inversion of the model the leaf biochemical content can be estimated from its spectral response. Similar relations were found between the optical properties and leaf chlorophyll content (Yamada & Fujimura, 1991). Leaf colour measurements provide a fast, cheap, and quantitative method of evaluation of the leaf nutrient status (Buscaglia & Varco, 2002). Reflectance measurements have shown to be a useful tool to identify the nutritional status of different plant species, said Graeff & Claupein (2003). Espagňa-Boquera et al., (2006) found spectral answer to the leaf N content, but a significant correlation was found only in the youngest leaves. In this paper we examined the effect of the growing site on the optical spectrum of the leaves of different strawberry varieties. Moreover the correlation between the leaf colour measured at flowering time and the fruit quality was investigated. Our aim was to find those features of the optical spectra that give information on the nutrition status of the strawberry plant.


Introduction
Strawberry (Fragaria x ananassa Duch.) is very important small fruit in Hungary. Strawberry is one of the earliest fresh fruits after the wintertime poor fruit supply in the market, and a delicious fruit not only for finished products, but also for ingredients to be included in complex foods such as ice-creams, cereals, dairy, confectionery, and bakery products (Dermesonlouoglou, 2006). The deep frozen products, jam, and preserved fruit are also popular.
Breeders have been producing several strawberry varieties with a broad range of capabilities. The most important qualities are the good level of resistance to Phytophtora cactorum, Ph. fragariae, Botrytis cinerae, and other strawberry diseases, high fruit quality, regular shape, long shelf-life, good smell and flavour, and attractive colour (Roudeillac, 1993). The different varieties, however, react differently to environmental and growing conditions.
Traditionally the evaluation of the yield of the plant is based on measurements of the fruit size and quantity and on organoleptic evaluation of the fruit quality (Nestby, 1998, Miner et al., 1997, Darbellay et al., 2000. However, this is a tedious process, moreover sensory evaluation always has a certain amount of subjectivity. In an extensive statistical evaluation Jacquemoud et al., (1995) investigated the correlation of the biochemical properties of 73 plant leaves of different species with their optical properties. They found good prediction performances for the protein, cellulose, and lignin content. Based on these correlations, radiative transfer models (Jacquemoud et al., 1996) were constructed which make it possible to calculate the simulated spectral response of a leaf model. Through the inversion of the model the leaf biochemical content can be estimated from its spectral response. Similar relations were found between the optical properties and leaf chlorophyll content (Yamada & Fujimura, 1991).
Leaf colour measurements provide a fast, cheap, and quantitative method of evaluation of the leaf nutrient status (Buscaglia & Varco, 2002). Reflectance measurements have shown to be a useful tool to identify the nutritional status of different plant species, said Graeff & Claupein (2003). Espagňa-Boquera et al., (2006) found spectral answer to the leaf N content, but a significant correlation was found only in the youngest leaves.
In this paper we examined the effect of the growing site on the optical spectrum of the leaves of different strawberry varieties. Moreover the correlation between the leaf colour measured at flowering time and the fruit quality was investigated. Our aim was to find those features of the optical spectra that give information on the nutrition status of the strawberry plant.

Materials and methods
We measured 6 strawberry cultivar`s leaf colour and nutrient content from 3 different growing areas. The growing system was the same for all places: raised bed, twin row, covered with black woven plastic covers and drip irrigation system between the twin rows.
The ecological conditions of the 3 places are rather different, concerning both the soil parameters and the climatic conditions. The three places: Pölöske is situated in West-Hungary among the Zala-hills. The climate is moderately cold -moderately humid. The sunshine duration is 1900 hours. The annual mean temperature is 9,5 ºC, the summer mean temperature is 15,5 ºC. The summer mean of the absolute maximum temperature is 32,5 ºC, winter mean of absolute minimal is -17,5 ºC. The annual precipitation amount is about 700 mm/year. In summer: 440-450 mm. The type of the soil is clay brown forest soil. Kecskemét is situated in the middle of Hungary in the Alföld. The climate is moderately hot -dry. The sunshine duration is a little bit under 2100 hours. The annual mean temperature is 10,2-10,4 ºC. The summer mean of absolute maximum temperature is 34,2-34,5 ºC, in winter mean of absolute minimal is between -16,5 and -16,7 ºC. The annual precipitation amount is about 540-550 mm/year. In summer: 310-320 mm. The type of soil is light lime sandy soil. Újfehértó is situated in Eastern-Hungary called Nyírség. The climate is moderately hot (but near the moderately cold) -dry. The sunshine duration is nearly 2000 hours. The annual mean temperature is 9,5-9,6 ºC. The summer mean of the absolute maximum temperature is about 34 ºC, in winter mean of the absolute minimal temperature is -17 ºC. The annual precipitation amount is about 560-590 mm/year. In summer: 350 mm. The soil type is acidic sandy soil (Marosi et al., 1990).
We have collected leaf, and fruit samples. Leaves: The collected samples were most -recentlymatured leaves -those leaves that have reached maximum size, i.e. essentially stopped expanding size. These leaves also have changed from a juvenile light green colour to a dark green colour, which is very important to colour measuring. We collected the samples at flowering time. We picked 5 leaves per plant, measured their colour and the nutrient content: nitrogen, phosphorus and potassium. The nutrient content was measured in laboratory by standard chemical analysis techniques.
The colour of the leaves was measured by a MINOLTA-200 colorimeter.
This handheld instrument contains a built in light source and measures the reflection of the sample. Primary data are represented as CIE (Commission Internationale de l'Eclairage) tristimulus (X, Y, Z) values (MacAdam, 1981, Wyszecki & Stiles, 1982, where the sensitivity curves of the X, Y, and Z detectors match closely that of the red, green, and blue sensitive cones of the human eye. We calculated the λ d dominant wavelength from the measured tristimulus values in the following way. First the XYZ -> L*a*b* -> LCH transformation was performed using standard procedures, where L is the lightness value, C is the chromaticity parameter, and H is the hue of the colour. The λ d dominant wavelength associated to the measured tristimulus values is defined as the wavelength of that pure colour (i.e. a spectral distribution having an infinitely sharp peak at λ d ) which gives the same hue value as those measured, i.e. it is given as the solution of the following equation: The λ d solution of this nonlinear equation was computed numerically for each measured H leaf value (MINOLTA CR-200).
All calculated λ d values lie between the 499 nm (green) and 578 nm (yellow) values. In this wavelength range Eq. (1) can be linearized with a good approximation (R 2 =0,9975), (2) The introduction of the dominant wavelength has several advantages. On the one hand while the tristimulus values are "antropomorph" by their very definition -matching the sensitivity of the human eye -calculating the dominant wavelength this antropomorph factor is "transformed out" in some extent, because the wavelength is a physical parameter of the light. On the other hand the use of λ d makes us possible to M. Deák, Sz., Márk, G. I., Szabó, T. & Füleky, Gy.  Fruits: The fruit samples were collected from the second and third picking. After the picking the berries were kept in deep fridge at -20 ºC. Before the procedure of sensory examination, the samples were put in a 10% sugar solution.
The taste panel stood about 18-20 persons. At the judging four parameters were estimated: size and form, colour, flavoursmell, and fruit firmness (Códex, 2000). The characters were graded between 1-5 point (with a possibility of giving half a point). Dirinck et al., (1981. ) examined strawberry fruits with similar methods but on fresh fruit and between 1-9 value. Darbellay (2000) and Wozniak et al., (1996) have used a similar method to valuate the strawberry varieties' quality.
By its very sensory nature organoleptic examination does not give absolute, but only relative results for the judged parameters. This means that only the differences between the ranks given during the same judgement can be taken into account. Since the samples from the 3 growing sites were evaluated in 3 different judgements (but partly by the same persons) a special data procession was necessary in order to compare the results. To this end the variety Elsanta was chosen as a standard variety and the grade differences relative to Elsanta were calculated.
The data processing was made by ANOVA method (Sváb, 1965). Figure 1 shows the (Y, λ d ) colour parameters (lightness and hue) of the leaves as a function of the growing site and the variety. The upper row shows the Y lightness parameters and the lower row shows the λ d hue parameters, the numerical values are given in Table 1, and 3. a(d). shows how the lightness (hue) values change for each variety for the 3 places. b(e) shows the same data grouped by the site -this shows the variation of the colour at each particular growing site. We can see that while at Újfehértó the colour of the varieties shows only a small scattering, at Kecskemét, however, the colour of the different varieties have a considerable scattering. This is further demonstrated at c(f) where the average and the standard deviation of the lightness (hue) parameters are displayed (Table 1) as the function of growing site.

Leaf colour measurements
As can be seen in Tables 2 and 4, both the effect of the variety and site are significant at 0.1% level. The interaction of the two factors is also significant at this level.
Though both the variety and the growing site does have a significant effect on the Y and λ d colour parameters, it is also important to know, for which particular pairs of variety and for which particular pairs of the growing site the difference of the measured Y and λ d colour parameters is significant. This is accomplished by comparing the Y vi -Y vj , Y ai -Y aj , λ viλ vj , and λ aiλ aj differences with the corresponding SD 5% levels (cf. Table 2. and Table 4.), where "vi" and "vj" are the variety index (1 for Spaeka, 2 for Raurica, …, and 6 for Camarosa) and "ai" and "aj" are the growing site index (1 for Újfehértó, 2 for Pölöske, and 3 for Kecskemét). If the absolute value of the difference is greater than the corresponding SD 5% level, i. e. their quotient is greater than one, then the corresponding difference is significant. Tables 5, 6, 7, and 8 show the results of this analysis as triangular matrices. (The sign of the difference changes when the two indices are exchanged, e.g. (λ viλ vj ) = -(λ vjλ vi ),  hence the lower part of the matrix would not give us additional information.) A number "1.0" means that the corresponding difference is equal to SD 5% . Numbers less than one mean that the difference of the given factor pair is not significant. Numbers greater than one (enhanced with grey background on the tables) mean that the difference is significant. Greater numbers denote a larger significance of the given factor pair We can see in Tables 5 and 7 that the effect of the growing site on both the lightness and the dominant wavelength parameter is significant for all site pairs but the significance is much greater when one of the sites if Kecskemét in the comparison.

Spectral properties of strawberry plants
Tables 6 and 8 show that the effect of the variety is not significant for all combinations and the growing site has a marked effect on how distinguishable are the varieties. (This can also be seen on Tables 2 and 4 which show that the interaction of the "site" and "variety" factors if also significant).
At Újfehértó, there are no significant differences between the colours of the varieties (this is true for the lightness and for the dominant wavelength). At Pölöske, however, we can see already some significant differences. Concerning the lightness factor, the variety Spadeka is distinguishable from Marianna and Camarosa in its lightness. We can see in Table 8 that the dominant wavelength difference matrix in Pölöske is a block matrix, i.e. if we divide the 6 varieties into two groups: Spadeka, Raurica, Symphony and Elsanta, Marianna, Camarosa, then we can observe no significant differences in λ d within the two groups but the inter group differences are significant. At growing site Kecskemét there are significant colour differences in between all varieties but the Raurica-Symphony pair in lightness and the Symphony-Elsanta pair in λ d .

Sensory examination
Now we turn to the investigation of the sensory examination results. As we explained above, the sensory results can be "normalized" by recording the grade differences relative to a standard variety. We choose Elsanta for this purpose. In order to study the effect of the growing site we calculated the Pölöske-Újfehértó and Kecskemét-Újfehértó grade differences, i.e. Table 9 shows how the flavour-smell grade of a given variety changes at the different growing areas.
If we compare the such calculated Kecskemét-Újfehértó flavour-smell grade differences to the measured leaf lightness at Kecskemét, Figure 2, a very good correlation is seen between these two quantities. As concerns the Pölöske-Újfehértó difference and the other sensory parameters, smaller correlation coefficients were found.

Discussion
Our leaf colour measurements show that in optimal ecological and nutritional conditions the leaf colour of the different strawberry cultivars shows only a small variation -the M. Deák, Sz., Márk, G. I., Szabó, T. & Füleky, Gy. Table 6 Significance levels of the "variety" factor pairs on the "lightness" colour parameter. Significant differences are shown with a grey background. We choose to characterise the lightness of the leaves with the Y tristimulus component because of two reasons. First, the lightness L is calculated in the L*a*b* colour system from Y as L = 116 (Y/Yr) 1/3 -16 , and second, we found it more suitable to use Y instead of Y 1/3 for our purposes because this nonlinear transformation improved the significance levels in the ANOVA calculation. The dominant wavelength measurements of Pölöske leaves ( Table 8) show that the 6 cultivars can be divided into two groups: Spadeka-Raurica-Symphony and Elsanta-Marianna-Camarosa. We observed no significant intragroup colour differences and significant inter-group differences.

Újfehértó
We found a very good correlation ( Figure 2) between the measured leaf colour at growing site Kecskemét and the result of the sensory examination for the flavour-smell of the fruit. This significant result makes a connection between an objective measurement performed by an instrument and an average of sensory examination results subjectively given by a panel of observers.
The genetically determined leaf colour depends on the optimal nutrition (Ulrich, 1980). Our soil analysis results showed (Table 10) high P and K levels in Kecskemét and these high levels prevented the plant to take up the other nutrient elements which caused the discolouration of the leaves and the poorer flavour of the fruit.

Conclusions
Finally we can say that the effect of the growing area is very important because it makes a strong influence to the colour of the strawberry leaves. At the places where the circumstances are not optimal for the plants, their leaves' Table 8 Significance levels of the "variety" factor pairs on the "dominant wavelength" colour parameter.

Spectral properties of strawberry plants
Significant differences are shown with a grey background.  Table 9 Organoleptic flavour-smell differences for Pölöske-Újfehértó and Kecskemét-Újfehértó, relative to Elsanta.  colour has greater differences than in case of an optimal place. By careful study of the sensory examination data we have found that the Újfehértó growing site was the best for the examined strawberries, Pölöske was optimal either, and in Kecskemét we found a strong negative correlation between the leaf colour and the flavour of the fruit. If the leaf colour of the strawberry plant is lighter and more yellow in flowering time, it anticipates a degradation of the fruit flavour. This result makes it possible to foresee the fruit quality based on simple, but objective leaf colour measurements. A strawberry plant with unsatisfactory nutrient supply can not produce the genetically determined fruit quality. The unsatisfactory nutrient supply causes the discoloration on the plant leaf which is measurable by the colorimeter. The more yellow the leaf the less good tasty the fruit is.

Spadeka
When the light green or yellow-green leaves are visible on the plants at flowering time, there is still possibility to intervene and change the nutrient supply, in order to improve the strawberry fruit quality. This paper presents the first result of a complex experiment with three different growing areas and 6 strawberry varieties, and different nutrient supplies. Results for the connection of the nutrient content and leaf colour, and fruit quality will be published later.