Effect of Cane Vigour on the Kiwifruit Quality of A. Deliciosa and A. Arguta

Kiwifruit has not been studied as much as other well-known fruits especially when it comes to studies about plant vigour and training systems. The aim of the study was to determine the importance of cane vigour of Actinidia deliciosa 'Hayward' and Actinidia arguta 'Issai' in order to develop the proper pruning technique that results in the best fruit quality. In addition, the effect of storage parameters such as weight, rmness and quality of the fruit was also studied. The study showed that the fruit size and weight are lower in low vigour canes in A. arguta, in contrast to A. deliciosa, where the fruit size and weight are smaller on high-vigorous canes. For A. arguta, it is recommended to choose high-vigour canes as the optimal fruit wood during pruning. In this way, the fruits will ripen more evenly. The other possibility is to perform the harvest two to three times per season to achieve a more uniform fruit quality. In the case of A. deliciosa the fruit are less variable between different cane vigour, so harvesting can be done in a single picking. In A. deliciosa the less vigorous canes tend to show a slightly better fruit quality.


Introduction
Commercial production of kiwifruit started in New Zealand in 1930, and in 1960 the rst kiwi orchards were established in Europe, especially in Italy and France. The Actinidia species comprise 76 species and 120 subspecies, of which only three are economically important for kiwifruit production: A. chinensis, A. deliciosa, A. arguta and their hybrid cultivars 1 . Due to this short period of commercial production, not a lot is known about these fruit species 2 .
The most commonly grown variety is A. deliciosa 'Hayward'. The fruits of A. arguta are quite different from those of A. deliciosa, they are smaller, not covered with trichomes, so we can eat them whole without peeling the skin 1,3 .
For commercial production, the economy of fruit production is of crucial importance. Higher vigour canes produce higher number of fruits, because of the higher number of winter buds and consequently higher number of burst buds as well as higher carbohydrate availability 4 . Fruits are supposed to be larger on longer canes and closer to the base of the cane 5 .
Kiwifruit are generally harvested at the technological stage of fruit maturity, while fruit is still hard and not ready for consumption. The time of picking is usually determined by measurements of rmness, total soluble solids and dry matter 6 . During storage, parameters such as kiwifruit weight, rmness, sugars, organic acids and total phenols change. Fruit weight, rmness, organic acids decrease, while sugars and total phenols increase 7 . Storage is very important as it affects the organoleptic properties and taste of the fruit. A. deliciosa 'Hayward' can be stored for up to 6 months at a temperature between 0 and 1°C 8 , while varieties of A. arguta can be stored for up to 28 days 9 at a temperature between 1 and 2°C 10 .
Fruits containing more sugars are generally more sought after by consumers. Unripe fruits of A. deliciosa contain less total and individual sugars than ripe fruits 3,11,12,13,14 . A. deliciosa and A. arguta differ both in their total sugar content and in the proportion of individual sugars. Fruits of A. deliciosa contain about equal shares of glucose and fructose as well as only up to 15% of sucrose 3,11,12 while in fruits of A.
arguta the share of sucrose can be up to 80% 3,15 . The difference in the sucrose content of fruits between the two species could be explained by the lower invertase activity in the last ripening stages and the higher sucrose phosphate synthase activity in fruits of A. arguta compared to A. deliciosa 16 . Total sugars increase during ripening and after storage, but the ratio between the individual sugars remains the same 17 .
Besides sugars, organic acids are the most important compounds that affect the fruit taste. They give the fruit an indistinct avour, as well as reduce bacterial infections that spoil the fruit 3 . The fruits of A. deliciosa contain equal shares of citric acid and quinic acid and only up to 10% of malic acid 3,11,17 , while the fruits of A. arguta citric acid is predominant followed by quinic acid and malic acid 3 . Organic acids remain at the same level or decrease slightly after storage 17 .
Phenolic compounds are secondary metabolites, plants synthesise them during development in response to stress. The most common group of phenolic compounds are avonoids, which are widely distributed in the plant and have many biochemical and pharmacological effects that have a positive impact on human health 18 . Phenolic content in the kiwi fruit is highest in the protective peel, in A. arguta up to 15 times higher than in the pericarp. Since we eat fruits of A. arguta whole, without peeling, the amount consumed is much higher than in A. deliciosa varieties, up to three times higher in commercially important varieties. A high phenolic content, combined with vitamin C, is usually associated with the antioxidant effect of the fruit 19 .Storage has a positive effect on the total phenolic content of the kiwi, due to the changes in phenolic metabolism and the higher activity of the PAL enzyme 20 . Therefore, the total phenolic content after storage usually increases from 5 to 10 mg per 100 g fresh weight after 4 months storage at 0 °C 13 .
The main focus of this study is to nd out how different cane vigour of A. deliciosa and A. arguta affects the fruit size and the fruit quality. Work in terms of determining the best canes for the highest fruit quality was carried out in order to re-evaluate the pruning techniques to con rm or modify the pruning guidelines for A. deliciosa and A. arguta. Considering the previous work in the eld, we expected lower vigour canes to produce smaller, lighter fruit of lower fruit quality than higher vigorous canes. It was expected that after storage, the total and individual sugar content in the fruits would be higher and the total and individual content of organic acids, total phenols and rmness would be lower. Only fruit of A. deliciosa was measured after storage, as fruit commercially stores up to 6 months under the right conditions, while fruit of A. arguta does not store well and is usually put on the market immediately after harvesting.

Plant material
The trial was carried out on A. deliciosa 'Hayward' and A. arguta 'Issai' in 11-year-old kiwifruit orchard, located in Štatenberg, Makole, Slovenia (altitude 323 m; 46° 20' 02" N; 15° 39' 32" E). All the analyses (fruit weight, size, rmness, total and individual sugars, total and individual organic acids and total phenolic compounds) were analysed in a laboratory of Biotechnical faculty, Department of Agronomy located in Ljubljana, Slovenia.
The trial was established after winter punning 2018. On 20 th of February (BBCH 00) for each species, 30 canes were selected and pruned to a length of 40-50 cm. For each cane diameter was measured 1 cm from the base of the cane and the canes were divided into 3 categories (Table 1). On 10 th of May (BBCH 55) the owers were manually thinned, leaving only the main owers, full blooming appeared on 25 th of May (BBCH 65), further on manual thinning of fruit was done on 24 th of June (BBCH 74) to set the fruit load on each individual cane uniformly. The manual thinning of the fruits was carried out in such a way that each annual shoot had 1 fruit on low vigour cane, 2 fruits on medium vigour canes and 3 fruit on high vigour canes, both on A. deliciosa and A. arguta. The total number of fruits examined for their quality was 60 per species, proportionally from different type of cane vigour.

Sample preparation
The samples were prepared according to Mikulic-Petkovsek et al. (2007). The extract for sugars and acids was prepared from 5 g of fruit pericarp without the peel of A. deliciosa and 5 g of whole fruit of A. arguta, as the fruits of A. arguta are eaten without peeling and the fruits of A. deliciosa must be peeled before consumption. Plant material was cut into small pieces and 25 ml of double distilled water was added to the test tubes of each sample. Samples were placed on a shaker for 30 minutes on room temperature. After they were put in a centrifuge (Eppendorf Centrifuge 5810 R) for 5 minutes at 5000 rpm at 4 °C.
Samples were ltered through a cellulose lter Chroma l A-20/25 produced by Macherey-Nagel (Düren, Germany), transferred to a vial and stored at -20 °C until analysed by high-performance liquid chromatography (HPLC).
Extract for phenols was prepared according to Mikulic-Petkovsek et al. (2010) in test tubes from 5g of fruit pericarp without peel of A. deliciosa and 5 g of whole fruit of A. arguta, and 10 ml of methanol with 3% formic acid was added. Test tubes were places in a cooled ultrasonic bath for 60 minutes. After the sample was centrifuged for 8 minutes at 8000 rpm, supernatant was ltered through a polyamide lter Chroma l AO-20/25 produced by Macherey-Nagel (Düren, Germany), transferred to a vial and stored at -20 °C until the start of the analysis.

Analytical methods
Primary metabolites were analysed using a HPLC system connected to a RI plus detector (Finnigan Surveyor, Thermo, San Jose, USA) for determination of individual sugars and a diode array detector Organic acids were determined with the help of external standards for citric, quinic and malic acid (Fluka Chemie GmBH, Buchs, Switzerland).
Quanti cation was based on comparison of peak areas of samples with standard solutions. Content of individual sugars and organic acids were expressed in g/100 g of fresh weight (FW) of fruits.
UV/VIS spectrometer (Lambda Bio 20, Perkin Elmer, Waltham, USA) was used to determine the total phenolic content. Total phenolic content (TPC) of extracts was assessed by the Folin-Ciocalteau phenol reagent method (Singleton et al., 1999). For TPC a 10 ml test tube was used in which 7.9 ml of water was pipetted. After 100 µl of extract and 500 µl of Folin-Ciocalteau reagent was added. The extract was in (1:2 ratio (v/v) with MeOH). The samples were placed on a room temperature for 4 minutes, after 1.5 ml of sodium carbonate (20% w/v) and the remaining water was added so we had exactly 10 ml. The tube was closed, shaked and put in the oven on 40 °C for 30 minutes. A blind sample was prepared as well, which had 100 µl of methanol instead of the extract. The absorbance was measured at 765 nm, for each sample we had 3-repetitions. Total phenolic content of kiwi fruit was expressed as gallic acid equivalents (GAE) in mg/100 g FW of fruit.

Statistical analysis
Data was arranged in Microsoft Excel (Microsoft, Redmont, USA) and statistical analysed with R commander. For the determination of statistical differences between fruit, grown on canes with different vigour, one-way variance analysis (ANOVA) was used. The statistical differences between mean at 95 % con dence level were calculated. All data in tables shows average values with standard errors and statistical differences, marked with different letters.

Fruit size, weight and rmness by cane vigour
The cane vigour didn't affect the dynamics of fruit growth in either A. arguta or A. deliciosa, as shown in Table 2. In the rst measurements in A. arguta, the fruit was largest in high vigour canes and smallest in low vigour canes, the trend was maintained throughout the measurements and the difference was also statistically different. The same relationship was observed with fruit weight, as shown in Table 3, where differences between fruit weight and cane vigour were observed. In A. deliciosa the medium vigour canes bore the larger fruit compared to the high vigour canes. The fruit of A. arguta was the heaviest in high vigour canes, while the fruit of A. deliciosa was the heaviest on the low vigour canes ( Table 3). The results were not statistically different, except between the fruit from high vigour canes and the fruit from low/medium vigour canes in A. arguta. In A. arguta, the fruit from high vigour canes was 15.7 % heavier than the fruit from medium vigour canes and 20.5 % heavier than the fruit from low vigour canes. Even if the differences in fruit weight in A. deliciosa between fruit from low and high vigour canes were not statistically signi cant, the fruit from low vigour canes was 6.4 % heavier than the fruit from high vigour canes. The cane vigour affected fruit rmness in A. arguta, as shown in Table 4. The fruits of medium and high vigour canes produced the rmest fruit and low vigour canes the softest. The rmness did not statistically differ between the fruit of medium and high vigour canes, even thought, fruit from high vigour canes was for 23 % more rm than fruit from medium vigour canes. In A. deliciosa cane vigour didn't affect the fruit rmness, there was no statistical difference between canes of different vigour. In the storage period, the fruit rmness dropped substantially. Surprisingly, after storage rmness measurements revealed that the fruits of medium vigour canes remained rmer than the fruits of high vigour canes although the differences were not statistically signi cant.

Total and individual sugars
The main sugar in A. arguta was sucrose (63 %), followed by fructose (23 %) and glucose (14 %). In A. deliciosa, the main sugars after harvest were glucose (49 %) and fructose (43 %), followed by sucrose (8 %). The sugar content of both the individual sugars and the total sugars increased after storage, but the ratio between the individual sugars remained quite similar, with the main sugars being glucose (46 %) and fructose (47 %) followed by sucrose (7 %). The cane vigour affected total and individual sugar content in A. arguta, as shown in Table 5. The fruits of medium and high vigour canes produced the fruit with the lowest total as well as individual sugars, and fruit from low vigour canes the highest. Total sugars as well as individual did not statistically differ between the fruit of medium and high vigour canes, even thought, fruit from medium vigour canes had 9.4 % more total sugars, 13.6 % glucose, 5.6 % fructose and more 10 % sucrose than fruit from high vigour canes.
No clear in uence of cane vigour on individual and total sugars was noticed in A. deliciosa. High vigour canes produced fruit with a slightly lower sugar content, but the difference was minimal and not statistically signi cant. Storage for one month resulted in substantial increase in total and individual sugar. Surprisingly at after storage measurements, fruit from medium vigour canes seemed to contain more sugars, which were less variable than sugars in fruit from low, or high vigour canes, however this fact should be further evaluated.

Total and individual organic acids
There were no statistical differences between total and individual organic acids among treatments in neither A. arguta nor A. deliciosa, as shown in Table 6. Interestingly fruits from low/medium vigour canes of A. arguta had the highest organic acid values and opposite in A. deliciosa, the highest content of organic acids was observed in fruits of high/medium vigour canes, in both before and after storage measurements. The total organic acids in the fruits decreased after storage, with individual acids: citric acid decreased slightly, quinic acid remained at about the same level and malic acid increased slightly.

Total phenols
Total phenols (Table 7) were expressed in milligrams of gallic acid equivalents per 100 g of fresh fruit. There were no statistical differences between phenols and cane vigour in either A. arguta or A. deliciosa, but in both species a slight tendency could be observed that higher vigour canes produced fruit with higher phenolic content. Total phenols increased just slightly in fruit after storage.  Volz et al. (1991) and Thorp et al. (2003) reported that larger and heavier fruits were on the higher vigour canes and closer to the base of the cane. Our results for the fruits of A. arguta support these ndings, whereas the fruits of A. deliciosa do not show a clear picture, as there were no statistical differences in fruit weight between canes of different vigour, but only in fruit size. Heavier fruit in low vigour canes, but smaller in shape compared to the medium vigour canes could be a result of the imperfect fruit shape, as fruit was measured at the equatorial section at the minimum diameter of the fruit. The reason for two opposing results between the plants in the same genus could be of high importance in managing and pruning of the vines, as different techniques could apply in A. deliciosa and A. arguta. Firmness in storage acceptable kiwifruits of A. deliciosa 'Hayward' must be between 80 and 100 N 6 and that of edible fruit between 4 and 10 N 24,25 . Firmness in storage acceptable kiwifruits of A. arguta must be between 20 and 35 N and that of the edible fruit between 1.5 and 3.5 N 9 . In terms of fruit rmness higher vigour canes produce rmer fruit in A. arguta, the same, but not statistically signi cant, was observed in A. deliciosa. No studies are yet known with which we could compare the results.

Total and individual sugars by cane vigour
A. deliciosa and A. arguta differ not only in their total sugar content but also in their sugar composition. The proportion of glucose (49 %) and fructose (43 %) in A. deliciosa is much higher than that of sucrose (8 %). The results in fruits of A. arguta are similar to those of Nishiyama et al. (2008), who reported that the sucrose content in A. arguta is much higher than the glucose and fructose content. In our experiment, the sucrose content was 63 %, followed by fructose with 23% and glucose with 14%. The reason for the high sucrose content in A. arguta is due to lower invertase activity and increased sucrose phosphate synthase activity in the last stages of fruit ripening 16 . Jordan et al. (2000) reported that total sugars increase in storage as starch converts to glucose and fructose during the ripening of the kiwi fruit, which is consistent with our results where sugars in A. deliciosa increased after storage up to 3.6 times compared to the content at harvest. While the total sugar content increased, the ratio between individual stayed fairly the same which is in accordance with MacRae et al. (1989) and Amodio et al. (2007).
Fruits from A. deliciosa compared between different vigour canes had a similar sugar content, both before and after storage, while fruits from A. arguta differ quite a lot by different cane vigour. Fruits from low vigour canes had a 61 % higher total sugar content than fruits from medium and 88 % higher content compared to fruit from high vigour canes. This combined with the data on fruit rmness (Table 4) suggests that fruits from lower vigour canes ripen earlier than fruits from higher vigour canes. Based on the results, the picking of fruit in A. arguta is recommended to be done in 2 to 3 terms, taking the cane vigour into consideration.

Total and individual organic acids by cane vigour
In A. deliciosa, the organic acid ratio was 50 % citric acid, 40 % quinic acid and 10 % malic acid, which matches the results of Marsh et al. (2004) and are similar to Nishiyama et al. (2008) as well. Compared to A. deliciosa, the fruits of A. arguta have more citric (55 % of total acids) and malic acid (16 %) and less quinic acid (27%). In A. arguta the organic acid ratio was citric acid, quinic acid and malic acid, while Nishiyama et al. (2008) reported slightly higher citric and quinic acid and lower malic acid concentrations. Marsh et al. (2004) reported that after storage at 4 °C, citric acid decreases, malic acid content increases and quinic acid content remains the same, which is comparable to our results where after storage at 5 °C, citric acid decreases (for 12 %), malic acid content increases (for 17 %) and quinic acid content remains almost the same (>1 % decrease).
The cane vigour did not affect the total or individual organic acids in either A. arguta or A. deliciosa. Even thought there were no statistical differences, low vigour canes in A. arguta produced fruit with 4.5 % more total organic acids than high vigour canes. Whereas low vigour canes in A. deliciosa produced fruit with 4.5 % less total organic acids than medium and 5 % than high vigour canes.

Total phenols by cane vigour
The total phenols in A. arguta were 11-19.5 % higher than in the fruits of A. deliciosa, which contradicts the results of Leontowicz et al. (2015), in which A. arguta contains 2-3 times more phenols than A. deliciosa. The differences in phenolic content between both Actinidia species is probably due to the fact that in case of A. arguta phenolics were measure in pulp with the peel because the fruit is eaten whole, while fruit of A. deliciosa was pealed as the fruit is eaten without the peel. Peel is the outher barrier of the fruit and normally contains more phenols compared to pulp 26  Even if there was no statistical difference, higher vigour canes produced fruits with slightly more phenols in both A. deliciosa and A. arguta, therefore cane vigour could affect the total phenolic content, however further detailed studies should be conducted.

Conclusion
Interestingly, the fruits of A. deliciosa from medium vigour canes showed the least variability in sugar, organic acid and phenolic content after storage, which suggests that medium vigour canes produce the most uniform fruit thus minimising losses due to repackaging and uneven ripening. It appears that cane vigour does not affect the fruit quality, with the exception of the fruit shape.
In A. arguta, the fruit showed characteristically uneven ripening. Data on total and individual sugars and fruit rmness indicate that the fruits of A. arguta ripen earlier from lower vigour canes. In order to achieve more uniform ripe fruits, harvesting should be done in 2 or 3 terms, taking cane vigour into consideration.
With less variable fruit, storage could be better controlled and longer, as the fruit would ripen more evenly. With the right pruning techniques, better and longer market coverage could be achieved and losses due to repacking and uneven ripening would be minimised.
The lack of research, particularly on the effect of cane vigour on kiwi fruit quality and work on hardy kiwifruit (A. arguta), has been challenging, so pioneering work has been done in determining cane vigour and fruit load and its effect on fruit quality. The present study provides interesting data for further work on pruning, training systems and fruit quality for different Actinidia.