ISSN 1580-8432
ricultura
Vol 12, No 1-2	December 2015
Published by the University of Maribor, Faculty of Agriculture and Life Sciences, Slovenia
Vol 12,No 1-2
A
December 2015
gricultura
Editor-in-Chief
Dejan ŠKORJANC, University of Maribor, Slovenia
Edttorial Board
Adriano CIANI University of Perugia, Italy
Alea GASPARIC Krka, Slovenia
Andrea ALBERA Associazione Nazionale Allevatori Bovini Razza Piemontese, Italy
Anton IVANCIC University of Maribor, Slovenia
Bto ENDONG Nanjing Agricultural University, P.R.China
Bruno BIAVATI University of Bologna, Italy
Ana POSPIS IL University of Zagreb, Croatia
Ernst RÜHL Forschungsanstalt Geisenheim, Germany
Fabio CAPORALI Universita della Tuscia, Italy
Franc BAVEC University of Maribor, Slovenia
Franci CUS University of Maribor, Slovenia
Thorsten HAASE Universität Kassel, Germany
Frans SWANEPOEL University of Pretoria, South Africa
Sylvie GRANGER Département des Sciences et Techniques Agronomiques, France
Hana WEINGARTL Nstional Center for Foreign Animal Disease, Canadian Science Center for Animal and Human Health, Canada
Igor VOJTIC Veterinary Administration of the Republic of Slovenia
tgor ZIBERNA University of Maribor, Slovenia
Irena RAJC AN University of Guelph, Canada
Jean-Fsançois ELÉOUËT Institut Nadional de la Recherche Agronomique, France
Sernej TURK University of Maribor, Slovenia
Ljubinko STARCEVIC University of Novi Sad, Serbia
Marc ROUX ENESAD, Département des Sciences et Techniques Agronomiques, France
Milan HOCEVAR University of Ljubljana, Slovenia
Amarendra Narayan MISRA Center for Life Sciences, School of Natural Sciences, Central University of Jharkhand, India
Nikolae KARITHONOV State Agraria University, Ukraine
Peter DO VC University of Ljubljana, Slovenia
Silvio KOSUTIC University of Zagreb, Croatia
Sonja JOVANOVAC University of Osijek, Croatia
SSefan VOGEL Universität für Bodenkultur, Austria
TeePUTMAN University of Alberta, Canada
Tito ZeMBREK University of Zagreb, Croatia
V. P. SKULACHEV Moscow State University, Russia
ViktorJEJCIC Agriculture Institute of Slovenia, Slovenia
Vjekoslav PAR University of Zagreb, Croatia
Zlatko CMELIK University of Zagreb, Croatia
Marjeta CANDEK POTOKAR Agricultural Institute of Slovenia Univesity of Maribor, Slovenia
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Vol 12, No 1-2
December 2015
ricultura
Contents
ARTICLES	Page
Business plan for a Zen garden
Maja ŽIBRAT, Karmen PAŽEK and Vesna WEINGERL.......................................................................................1-7
Antioxidant defences of Norway spruce bark against bark beetles and its associated blue-stain fungus
Mateja FELICIJAN, Metka NOVAK, Nada KRAŠEVEC and Andreja URBANEK KRAJNC............................9-18
The accuracy of the germination rate of seeds based on image processing and artificial neural networks
Uroš ŠKRUBEJ, Črtomir ROZMAN and Denis STAJNKO................................................................................. 19-24
Impact of various types of anti-hail nets on light exposure in orchards and quality parameters of apples- a rewiev Marinka BRGLEZ SEVER, Stanislav TOJNKO and Tatjana UNUK.................................................................25-31
Sweet maize growth and yield response to organic and mineral fertilizers, N rates and soil water regimes
Franc BAVEC, Martina BAVEC, Silva GROBELNIK MLAKAR and Milojka FEKONJA...............................33-40
Variations in leaf total protein, phenolic and thiol contents amongst old varieties of mulberry from the Gorizia region
Tina UGULIN, Tamas BAKONYI, Rebeka BERČIČ and Andreja URBANEK KRAJNC................................41-47
Agricultura 12: No 1-2:1-7 (2015)
DE
G
DE GRUYTER
OPEN
Business plan for a Zen garden
Maja ŽIBRAT, Karmen PAŽEK, Vesna WEINGERL*
University of Maribor, Faculty of Agriculture and Life sciences, Pivola 10, SI-2311 Hoče, Slovenia
ABSTRACT
The placement of a theme park in the form of a Zen garden, as a business opportunity in the Slovenian rural area, is discussed. The design of the garden, with all the major points of a standard business plan, is accurately presented, with a description of the business, branch, and services, market analysis, marketing strategy, financial projections, and a plan of the work and activities. The financial aspect is presented as the amount of investment, net present value, and internal rate of return. The amount of investment is estimated at €14.891, which should be reimbursed within 4 years of operations. The estimated internal rate of return is estimated at 16.86%. Part of the study is the market analysis - conduction of a survey into knowledge of, and interest in, Zen and Zen gardens. The principles of landscape ecology are respected, as the Zen garden would be set in the woods and will blend seamlessly into the landscape.
Key words: theme parks, sustainable tourism, investment, financial indicators, landscape ecology
INTRODUCTION
The Government of the Republic of Slovenia defines tourism as one of the most important economic and strategic sectors, as it creates new jobs and has a highly positive impact on balanced regional development. From the Slovenian Tourism Development Strategy (2012-2016), which is the basic document for planning of tourism development at the national level, we can see that the designers of Slovenian tourism have, in recent years, reached a consensus that all tourism development must become sustainable (Ministry of Economic Development and Technology 2012).
Sustainable tourism development does not deplete natural resources, and does not pollute the environment, while respecting culture and human and ethical values. We can talk about soft tourism, which is the most suitable for rural areas. At the same time, sustainable development focuses not only on environmental issues, but emphasises development that meets the needs of the present without compromising the ability of future generations. Sustainable development links the concern for the capacity of natural systems with the social challenges facing humanity. Tourism is one of the fundamental pillars of balanced economic, social, ethical,
and environmental development - these are the components that comprise sustainable development. Thus, tourism is becoming an extremely important sector of both the global and the local economy. The sector is expected to grow constantly, both in qualitative and quantitative terms.
Any development of tourism cannot happen without affecting the development of the entire region. When we attract tourists with one attraction, they will visit by the way also other attractions, they will use public transport, eat in local restaurants and sleep in nearby hotels, which brings benefit to all actors and hence the entire region. However, there may cause also negative effects, such as increased traffic and thus greater noise and air pollution.
Nowadays, "relaxed tourism" is booming as people are in a constant hurry and are under stress, so there is a growing demand for relaxation techniques and a need for places where they can find peace. In the north-east of Slovenia alone, there are 7 thermal spas, over 25 tourist farms, and many other activities which attract tourists who want on-site facilities.
The landscape is a functional set of ecosystems and their environment, which is namely open, but able to be to a certain extent self-regulating. Landscape ecology is the study of the composition, structure and function of landscapes. A
*Correspondence to: E-mail: vesna.weingerl@um.si
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Business plan for a Zen garden
landscape is not necessarily defined by its size; rather, it is defined by an interacting mosaic of elements (e.g. ecosystems) relevant to some phenomenon under consideration (at any scale). Thus, a landscape is simply an area of land (at any scale) containing an interesting pattern that affects and is affected by an ecological process of interest (Turner et al. 2001, Turner, 2005).
Zen is the way of thinking, striving for inner peace and acceptance of other people and things as they are. To live in the present, disregarding the past and do not worry about the future. During the development of the Zen, various teachers used their own methods and approaches and that's why Zen parts for Japanese and Chinese. In addition, Zen parts of the course also due to the geographical division of the two countries. The Buddhist concept is completely enclosed in a Zen only that this concept arose the legend of Siddhartha Gautama, nowadays known as Gautama Buddha. The great masters of Zen and Buddhism may precisely explain the similarities and differences. However, if you live according to the principles of Zen, you live also according to the principles of Buddhism and vice versa. The Japanese and the Chinese, who were the originators of Zen thinking, created special gardens, called Zen gardens, which use many natural materials, such as sand, stone, rocks, and bamboo. Simple lines are used that do not impose any form, so each visitor experiences the garden in his own way. Zen garden it can be used as a tool to facilitate the achievement of the Zen, or just for a walk, relax, enjoying the nature, or an idea how to arrange the home garden. In Zen gardens there are created simple lines that do not impose any form in order to cleanse the mind of the observer for the understanding of superfluous elements, and allow the viewer to easily achieve deeper insights (Zibrat 2015).
Creating a business plan is necessary in order to cover the economic aspect. The concept of "economy" encompasses the entirety of the economic sciences, which are experienced very rapid changes due to the diversity of research. The subject of the economy is in every human activity in which we are confronted with the problem of limited resources with which to achieve the stated objectives. The task of economics is to ultimately achieve maximum efficiency, or minimisation of costs (Znidarsic Krajnc,1995).
By definition, investment means "increasing, or at least maintaining the value of capital". The process of investment lies in the realization of the path from the idea or concept to the market. Property is a lever of investment, which requires a continued increase or maintaining of the value. When investing, the proceeds or output should always be higher than the input (Vuk 2001).
The aim of our study was to accurately present the plan and the company that we want to establish, and to assess the financial feasibility of the investment, which was estimated on the basis of the assessment of net present value and internal rate of return. Another important part of the study wasthe market research - the survey into the knowledge of, and interest in, Zen and Zen gardens.
MATERIALS AND METHODS
Net present value and internal rate of return
The preferred approach to the evaluation of investment projects is a dynamic method known as net present value analysis (NPV) (Pazek and Rozman 2008).
The two most important criteria for the selection of investment projects are net present value (NPV) and internal rate of return (IRR). Most often, projects are evaluated equally according to both criteria, but sometimes these two criteria offer different estimates. In theory, the concept of NPV is better, but IRR offers more results, and thereby solves the problem of the evaluation of more complex and multilayered projects. New equations of NPV contain all possible solutions for IRR as a component. The analysis confirms the theory that NPV is a better concept, but IRR remains a compulsory core part of the corresponding NPV.
The NPV estimates the effectiveness of investment in the present. NPV reduces the expected cash income from investments for the sum, which depends on three factors: the riskiness of investments, the expected inflation, and the desire of investors to get their money back upon withdrawal from the project. If the cash inflows exceed the amount of the investment, then NPV is positive, and the project is financially attractive for investors (Kierulff 2008).
The rule for an investment decision on the basis of NPV is that the investment can be taken if NPV is greater than 0, and rejected if the NPV is less than 0. If the NPV is equal to zero, the decision maker could decide to be indifferent. From the alternative investment options, we choose the one with the highest NPV. The investment is acceptable only in the case when there is no alternative investment, which would give higher value yields for the same cost (Cebokli 2011). For an investment of t periods, the formula is as follows:
Where: NPVt - Net Present Value (€), I - investment costs (€), TR - total revenue (€), TC - total costs (€), r - interest rate (%), t - time - number of years
The disadvantage of the NPV method is the fact that it does not take into account the value of creation of opportunities. Sometimes an investment that is inherently uneconomically feasible can create opportunities, which enable new investments for the company in favorable market conditions (Buckley 1998).
An associated concept is the IRR, which does not serve with nominal values, but rather with a discount rate, at which NPV = 0 and represents the maximum interest rate that would be transferred to an investment in the case of financing by credit.
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Business plan for 
Landscape ecology
Landscape ecology is a sub-discipline of ecology, which examines how patterns in space affect the spatial processes. It covers both the impact of biological processes on the landscape structure and the influence of the landscape structure on the wealth and distribution of organisms in space. The main concern of landscape ecology is the study of large-scale spatial heterogeneity, due to both natural and anthropogenic influences, and the effects of this heterogeneity on ecological processes and species persistence.
Setting a Zen garden in the woods is certainly an interference with the landscape, so the principles of landscape ecology need to be respected. Trees which absolutely have to be felled for the purpose of the garden arrangements will be used in the garden, to create simple benches, sculptures, and a variety of decorative accessories. For the purpose of the garden arrangements, it will also be necessary to fully clean the forest floor, which will destroy all undergrowth. To gain the required space, it will be necessary to remove the majority of young trees. However, it will not be necessary to discard them; they will be transplanted to the periphery, or used as an integral part of the garden. Some of the forest undergrowth plants - for example, various ferns and sedges - will be transplanted and arranged for the purpose of the systematic design of the garden.
Animals will not be driven out from the area of the garden. The assumption is that that they will eventually leave the area of their own accord, due to the noise. The area of the entire forest is much larger than that of the garden, so the animals will have the opportunity to withdraw to a quieter part of the forest.
A small market selling local produce, such as sausages, honey, pumpkin seed oil, teas, natural soaps, essential oils and so on, is planned to be in front of the garden. This will allow nearby residents to earn additional income, and, at the same time, will encourage the development of complementary activities in the region. Stands will be offered at a token rate (€2.00 per day).
Table 1: The estimated amount of investment required to bu
a Zen garden
The impact of tourism
Tourist activities are linked to the natural and cultural environment. An important part of Slovenian tourism is its abundant opportunities for recreation in an attractive natural environment with special and interesting elements. The expansion of different forms of recreation is increasing, due to people's desire to withdraw from urban centres and to enjoy active leisure pursuits in a healthy environment.
Increased interest in leisure activities in a natural environment brings with it a number of problems, including disturbance of the wildlife. This is known as environmental wildlife stress, and is caused by an increase in visitors and by the construction and maintenance of recreational facilities and infrastructures. Animals react to visual and auditory stimuli, as well as smells. In general, wildlife is better able to tolerate cyclical and repetitive stimuli than those which are unpredictable. For example, a motorway, which produces a constant background noise, is less disruptive to animals than a construction site which will produce sudden outbursts of noise at random times (Kolar 1999).
The development of tourism in a locality is not possible if it does not take into account the local population. It is important that local people see the value the development will add to the area. In order to gain their cooperation, local people should be integrated into the preparation of the development strategy, listened to, and, if possible, their opinions, suggestions and ideas should be taken in to account. In fact, the local population has a much greater sense of, and the best vision for, their needs for the development.
Any development of tourism cannot happen without affecting the development of the entire region. When we attract tourists with one attraction, they will visit by the way also other attractions, they will use public transport, eat in local restaurants and sleep in nearby hotels, which brings benefit to all actors and hence the entire region. However, there may cause also negative effects, such as increased traffic and thus greater noise and air pollution.
a Zen garden
	Amount	Unit	Price		Together (€)
Purchase of land	4,000	m2	1.00	€/m2	4,000.00
Paths (gravel)	30	m3	9.00	€/m3	270.00
Benches	15	pcs	80.00	€/pcs	1,200.00
Waste bins	10	pcs	30.00	€/pcs	300.00
Sand	15	m3	17.00	€/m3	255.00
Rocks	20	pcs	20.00	€/pcs	400.00
Vegetation	100	pcs	7.00	€/pcs	700.00
Machine work	50	h	30.00	€/h	1,500.00
Handwork	100	h	5.66	€/h	565.70
Website	1	pcs	500.00	€	500.00
Promotional material	2,000	pcs	0.10	€/pcs	200.00
Stands	5		500.00	€/stand	2,500.00
Project documentation					1,500.00
Other					1,000.00
				Total	14,890.70
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Business plan for a Zen garden
Business plan
A business plan is a detailed and measured description of the business or business idea. It is urgently needed if the entrepreneur wishes to obtain financial support from banks, to apply for financial aid from the various funds, or to expect cooperation from other companies. It must include real and measurable objectives, including a description of the business and branches, research and market analysis, marketing plan, financial analysis, risk assessment, and schedule. Financial projections are the most important element of any plan, and are formed after detailed analysis of the business, costs, and revenues. If the company already exists, the projections are based on results from previous years; if not, on forecasts for the future, usually for 3-5 years (Vidic 1999).
Zen garden
When designing a Zen garden, it is reasonable to bear feng shui in mind. Feng shui has been configured as a system that understands the multi-layered nature of space, together with its subtle energy levels. This is known as the school of forms, which talks about how energy flows through the landscape. The basic principle of the school of forms is that the landscape is a living being, which pervades the life energy "qi", and has the ability to regulate the flow of energy to be steady and balanced, with just the right level of intensity (Kryzanowski 2012).
In western countries, research has accumulatedon the therapeutic effects of nature since the 1970s. The gap between eastern and western countries on the topics of therapeutic landscapes and healing gardens bridges the common knowledge that green public spaces are beneficial to people's physical, mental and social health, by providing spaces for therapeutic activities and contemplation, which relieves stresses and encourages social communication (Jiang 2014).
Zen gardens seem to be an ideal place for meditation, as they are designed to defuse and concentrate thoughts. Therefore, the garden will have a lot of landscaped corners, which will offer visitors some privacy.
Zen gardening is an art form with a profound spiritual significance. Zen gardens are known as 'arid gardens' or 'stone gardens'. In their own language, the Japanese call them 'karesansui', which literally means 'water of dry mountains'.
Many Zen gardens are designed with the aim of symbolic communication of Buddhist concepts; it is possible to
experience 'Zen' in them, thanks to the particular vulnerability of nature to its most basic elements, which can still carry the whole message. For instance, the stone symbolizes a mountain, sand or curves in the sand the water, the piece of wood a tree, the forest a boat, and so on. The essence of such symbolism of communication is to cleanse the mind of the observer of all superfluous elements, and allow him to easily concentrate on his insight.
Table 3: Total operating costs of the Zen garden
	Nr	Net salary	Gross salary	Total per year	
Salaries of employees	1	995.64	1,521.38	18,256.60	€
Other costs					
Maintenance				1,200.00	€
Advertising				500.00	€
Total annual cost				19,956.60	€
Zen gardens sometimes include live plants in limited quantities, mostly moss, but always in proportion to the largely static and inanimate nature of such a garden. This is primarily a reflection of the Buddhist evaluation of the relative rarity of life and the cycle of birth and impermanence, which illustrates the change of seasons by the impact on the living nature in the garden. Many Zen gardens are positioned in such a way that each garden 'borrows' from its neighbourhood as part of its design. The purpose of such borrowing of the background is the optical zoom of the garden area and its logical connection with the surroundings. Planned gardens also fuse to the surroundings, so that they do not unduly interfere with the existing nature.
RESULTS AND DISCUSSION Investment costs
The investment is the largest financial outlay, but it is absolutely necessary. Table 1 shows the costs and total value of the investment. The highest cost is the purchase of land, amounting to €4,000.00.
Zen garden is planned in north-eastern Slovenia in the Pomurje region, where property prices are generally lower due to a lower standard of the whole region. We have assumed the approximate average price for forest land in the market €1/m2.
Part of the investment budget will be needed for machine work, buying stands, project documentation, and other aspects.
Annual cash flow
Cash flow is the movement of cash in and out during the period under analysis. This must be determined in order to
Table 2: Total revenue on an annual basis
	Price		Amount		Total	
Entrance fee	3.50	€	6,500	Visitors	22,750.00	€
Stands rental (5)	10	€/ day	262	Days	2,620.00	€
Total annual revenue					25,370.00	€
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Business plan for a Zen garden
ensure or assess the current solvency of the company. The annual cash flow (ACF) is the most important parameter when calculating the net present value. ACF was calculated as the difference between total revenue (TR) and total cost (TC). Table 2 presents the total revenues. The garden would be financed from ticket sales and the rental of stands.
The total costs, represented in Table 3, amount to €19,956.60. The biggest costs would be staff salaries. Initially, only one person will be employed at an average Slovenian salary for the month of June 2014, i.e. €1,521.38 gross. (SAOP 2014)
Other costs would be maintenance costs and advertising, which together account for €1,700.00 per year.
ACF = €25,370.00 - €19,956.60 ACF = €5,413.44
Break-even point of the business and breakeven price of tickets
The profitability threshold or break-even point is the moment when a company achieves a volume of business whereby it has neither a profit nor a loss. At such a volume, the company revenue sales just cover all costs. Above the breakeven price, the profit increases; below it, losses increase. A calculation of the break-even point of the volume of ticket sales, comprising the number of visitors and break-even price of tickets, is presented in Table 4.
Table 4: Break-even point in business volume
Nr. of visitors	Entrance fee	Revenues	Expenses	Profit
6,500	€3.50	€25,370.00	€19,956.56	€5,413.44
6,000	€3.50	€23,620.00	€19,956.56	€3,663.44
5,500	€3.50	€21,870.00	€19,956.56	€1,913.44
5,000	€3.50	€20,120.00	€19,956.56	€163.44
4,955	€3.50	€19,962.50	€19,956.56	€594
4,954	€3.50	€19,959.00	€19,956.56	€2.44
4,953	€3.50	€19,955.50	€19,956.56	€-1.06
With a planned entrance fee of €3.50, and an expected number of visitors of 6,500, the company would have a profit of €5,413.44. By reducing the number of visitors, the profit decreases. The minimum number of visitors with which the company would still operate positively, is 4,954; the profit would be €2.44.
By reducing the amount of the entrance fee, profit drops sharply.
The minimum ticket price, where the company would still operate positively, under the assumption that the expected number of visitors is achieved, is €2.70 (Table 5).
Table 5: Break-even price of tickets
Nr. of visitors	Entrance fee	Revenues	Expenses	Profit
6,500	€3.50	€25,370.00	€19,956.56	€5,413.44
6,500	€3.30	€24,070.00	€19,956.56	€4,113.44
6,500	€3.00	€22,120.00	€19,956.56	€2,163.44
6,500	€2.80	€20,820.00	€19,956.56	€863.44
6,500	€2.70	€20,170.00	€19,956.56	€213.44
6,500	€2.60	€19,520.00	€19,956.56	€-436.56
Net present value and internal rate of return
Evaluations of the net present value and internal rate of return are shown in Tables 6 and 7. The net present value was estimated by taking into account interest rates of 8.00%. Positive business would begin in the 4th year of operations; estimated NPV will by then be €3,039.30.
The maximum interest rate which can be managed with the investment is estimated at 16.86%; estimated NPV will then be €0.69.
Table 6: Assessment of the net present value after the 5th year of business
Year	Interest rate - 8.00 %	NPV (€)
1	5,012.44	-9,878.26
2	4,641.15	-5,237.11
3	4,297.36	-939.74
4	3,979.04	3,039.30
5	3,684.30	6,723.59
Table 7: Assessment of the internal rate of return		
Year	Interest rate - 16.86%	NPV (€)
1	4,632.41	-10,258.29
2	3,964.07	-6,294.22
3	3,392.15	-2,902.06
4	2,902.75	0.69
The survey
Among 136 respondents, there were 105 women and 31 men with an average age of 30-40 years, mostly from the countryside. Results showed that 52.2% of respondents preferred to spend their free time in natural surroundings. 36.8% of respondents were familiar with Zen and interested in the topic; 31.6% of respondents had not heard of Zen or a Zen garden, but they were interested in learning about it. 86.8% of respondents would visit a Zen garden if it was within a radius of 20-40 km. 26.5% of respondents did not mind how far away the attraction would be. 51.5% would visit a Zen garden in the springtime; 29.4% in the summer.
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Business plan for a Zen garden
68.4% of respondents intended to visit a Zen garden out of curiosity. In terms of the entrance fee, the majority (45.4%) were ready to pay €2-3; 30.1% were willing to pay €4-5. 80% of respondents already had a local market nearby, but only about 30% attended it. 89.7% of respondents preferred the possibility of purchasing local products or crops when visiting local attractions (Žibrat 2014).
CONCLUSIONS
The aim of the study was to accurately present the branch and the company that we want to establish, and to assess the financial viability of the investment, which was estimated on the basis of the calculation of net present value and internal rate of return.
The survey was included for the market research reasons. It was necessary to know whether the people are interested to see the Zen garden, whether they are willing to pay an entrance fee and how much. The results of the market research were positive.
Investment in the construction of a Zen garden with corresponding activities was financially justified. The investment of €14,890.70 would be repaid in the 4th year of operations, with a NPV of €3,039.30. In the case of financing by credit, investment may carry a 16.86% discount rate, which will encourage investors.
The investment would provide employees with a monthly income at the average Slovenian salary. As of June 2014, this was €1,521.38 gross; with the estimated visitor numbers, such a figure can be achieved. Moreover, the estimated investment clearly shows positive economic indicators; it is necessary to point out other benefits that tourism brings to the region. Development of tourism brings jobs and encourages investments in infrastructure and the preservation of natural and cultural heritage, which in turn raises the living standard of local people. Without investments, it is impossible to develop any branch. Alternatively, we may also expect impacts based solely on Zen garden, such as increasing interest in meditation, Zen, and/or yoga which is scientifically proven to have a positive impact on the quality of life of people who are engaged in this, as well as for the broader neighbourhood.
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Strategy of development of Slovenian tourism (20122016). http://www.mgrt.gov.si/fileadmin/mgrt.gov. si/pageuploads/turizem/Turizem-strategije_politike/ Strategija_turizem_sprejeto_7.6.2012.pdf (23 May 2014)
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assessment of business opportunities in spelt processing. Agricultura, 2008; 6: 13-17.
9.	SAOP d.o.o. http://www.saop.si/poslovne-informacije/ podatki-za-obracun-in-opomniki/povprecne-in-minimalne-place/ (3 September 2014).
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11.	Turner M.G. Landscape ecology: what is the state of the science? Ann. Rev. Ecol. Evol. Syst. 2005;36:319-44.
12.	Vidic F. Kako razviti uspešno podjetje. Gae College. Ljubljana, Slovenia. 1999.
13.	Vuk D. Gospodarjenje s tehničnimi sredstvi 2. Investicijski
management. Kranj, Moderna organizacija. Slovenia, 2001, 144p.
14.	Žnidaršič Krajnc A. Ekonomika podjetja. Postojna, Slovenia. 1994.
15.	Žibrat M. The survey: http://www.mojaanketa.si/ anketa/453511944/ (2014)
16.	Žibrat M. Poslovni načrt za zen vrt / The business plan for a Zen garden; diplomsko delo. Maribor. 2015. 37 p: https://dk.um.si/IzpisGradiva.php?id=48000(17. september 2015).
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Poslovni načrt za Zen vrt
IZVLEČEK
Diskutirana je umestitev tematskega parka - zen vrta, kot poslovna priložnost na območju slovenskega podeželja. Natančno je predstavljena zasnova vrta z vsemi glavnimi sklopi standardnega poslovnega načrta: opis podjetja in panoge, opis storitve, analiza trga, strategija trženja, finančne projekcije ter načrt dela in aktivnosti. Finančni vidik je predstavljen z oceno finančnih parametrov kot so višina investicije, neto sedanja vrednost in interna stopnja donosa. Višina investicije projekta je ocenjena na 14.890,70 €, povrnila naj bi se v 4 letih poslovanja. Ocenjena interna stopnja donosa ob povratku investicije je 16,86 %. Del raziskave predstavlja analiza trga -izvedba ankete o poznavanju in zanimanju za zen in zen vrt. Pri načrtovanju zen vrta so upoštevani principi krajinske ekologije, saj postavitev vrta v gozdvsekakor vpliva na krajino.
7
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Agricultura 12: No 1-2: 9-18 (2015)
DE
G
DE GRUYTER
OPEN
Antioxidant defences of Norway spruce bark against bark beetles
and its associated blue-stain fungus
Mateja FELICIJAN **, Metka NOVAK 2, Nada KRAŠEVEC 3, Andreja URBANEK KRAJNC 1
University of Maribor, Faculty of Agriculture and Life Sciences, Pivola 10, 2311 Hoče, Slovenia 2Kamlinska Institutet, Stockholm, Sweden National Institute of Chemistry, Ljubljana, Slovenia
SUMMARY
Bark beetles and their fungal associates are integral parts of forest ecosystems, the European spruce bark beetle (Ips typographus Linnaeus, 1758) and the associated pathogenic blue stain fungus Ceratocystis polonica (SIEM.) C. MOREAU, are the most devastating pests regarding Norway spruce [Picea abies (L.) H. KARST.]. Bark beetles commonly inhabit weakened and felled trees as well as vital trees. They cause physiological disorders in trees by destroying a phloem and cambium or interrupt the transpiration flow in the xylem. Conifers have a wide range of effective defence mechanisms that are based on the inner bark anatomy and physiological state of the tree. The basic function of bark defences is to protect the nutrient-and energy-rich phloem, the vital meristematic region of the vascular cambium, and the transpiration flow in the sapwood. The main area of defence mechanisms is secondary phloem, which is physically and chemically protected by polyphenolic parenchyma (PP) cells, sclerenchyma, calcium oxalate crystals and resin ducts. Conifer trunk pest resistance includes constitutive, inducible defences and acquired resistance. Both constitutive and inducible defences may deter beetle invasion, impede fungal growth and close entrance wounds. During a successful attack, systemic acquired resistance (SAR) becomes effective and represents a third defence strategy. It gradually develops throughout the plant and provides a systemic change within the whole tree's metabolism, which is maintained over a longer period of time. The broad range of defence mechanisms that contribute to the activation and utilisation of SAR, includes antioxidants and antioxidant enzymes, which are generally linked to the actions of reactive oxygen species (ROS). The presented review discusses the current knowledge on the antioxidant defence strategies of spruce inner bark against the bark beetle (Ips typographus) and associated blue stain fungus (Ceratocystis polonica).
Key words: antioxidants, ascorbate-glutathione system, blue-stain fungus Ceratocystis polonica (SIEM.) C. MOREAU, Norway spruce (Picea abies (L.) H. KARST.), phenolics, systemic acquired resistance (SAR)
THE BIOLOGY BEHIND THE ASSOCIATION OF SPRUCE BARK BEETLE WITH BLUE-STAIN FUNGI
Over their long lifetimes, conifers are targets of numerous attacks by different pests such as insects, other herbivores, fungi, and bacteria. They pave their way towards nutrient-rich phloem through the bark. Phloem and cambium represent a relatively small part of a tree's trunk, which could be even
faster and more easily damaged and destroyed than other tissues (Franceschi et al. 2005).
The majority of bark beetle species are considered as rather harmless species in their native ranges, colonising mainly weakened or dead trees and thereby represent an important ecological factor in forest rejuvenation (Müller and Job 2009, Smith et al. 2011, Novak et al. 2014). However, these species pose potential risks in the case of significant increase in abundance of populations and within changing or new environments. They should not be ignored when evaluating
Correspondence to: E-mail: mateja.felicijan@um.si
9
Antioxidant defences of Norway spruce bark against bark beetles and its associated blue-stain fungus
risks and threats to forest ecosystems or when determining quarantine measures for pests and pathogens. Forest pest insects and their associated micro-organisms are capable of movement through national boundaries and have a potential impact on forest-dependent industries (Christiansen and Bakke 1988, Linnakoski et al. 2012, Sallé et al. 2005, Raffa et al. 2008, Faccoli 2009, Smith et al. 2011).
One of the most destructive forest insects in Europe is the spruce bark beetle Ips typographus (Linnaeus 1758.) (Curculionidae, Scolytinae), which affects Norway spruce and is with its fungal associates, the pathogenic blue-stain fungus Ceratocystis polonica (SIEM.) C. MOREAU the integral parts of the forest ecosystems (Wermelinger 2004, Klepzig et al. 2009, Linnakoski et al. 2012, Novak et al. 2014). Their symbiotic relationship has been described as mutualistic (Whitney 1982). Although a number of studies have been devoted to resolving the nature of bark beetle-fungus interactions, these interactions remain poorly understood (Jurc 2011, Six and Wingfield 2011).
The fluctuations in bark beetles' p opulations are interactions between at least three main biological components: the host tree, bark beetles, and pathogen fungi (Novak 2014).
Normally has Ips typographus two (spring and summer) generations per year (Fig. 1). If global warming extends the growing season, a higher proportion of the second generation may reach the cold at the hardy adult stage and survive the winter (Holger et al. 2006). Bark beetles construct galleries under the bark within the phloem layers of woody plants, mostly in weakened or recently killed trees, where they lay eggs and their brood feed and develop during the winter (Six and Wingfield 2011).
Adult beetles form wounds through the bark into the phloem, as a source of food and the place of reproduction, to the cambium. Parental adults construct an initial egg gallery, which is continued by larvae into the larval galleries (Raffa et al. 2008). During the constructions of galleries, bark beetles serve as vectors for several types of micro-organisms that enter into the host tree. They benefit the fungus because it is transferred into a nutrient-rich environment, where competition is limited (Whitney 1982, Beaver 1989, Paine et al. 1997). Specific roles of fungal associates in the Ips typographus symbiotic system and some of its underlying molecular mechanisms have been the topics of considerable previous work (Krokene and Solheim 1998, Hammerbacher et al. 2013, Urbanek Krajnc et al. 2014).
Now it is known that within a symbiotic system bark beetles serve as vectors for fungi, which would otherwise be unable to reach a new host (Six and Wingfield 2011), alternatively fungi benefit the beetle as sources of nutrients, or they may weaken tree defences (Linnakoski et al. 2012). The concept that tree-killing bark beetles require fungal pathogens to overcome tree defences and to incur tree mortality has received the most attention over the years. This hypothesis, which is referred to as the classic paradigm, has formed the basis for the majority of research conducted on these interactions (Six and Wingfield 2011). Blue-stain fungi are thought to be primarily responsible (Johnson and Croteau 1987) or required (Whitney 1982) for mortality regarding conifers attacked by bark beetles.
Such conclusions are based on observations that:
-	beetles are capable of vectoring or dispersing the fungi,
-	beetles are rarely found in the absence of staining fungi,
-	sapwood of beetle-killed trees is stained (Paine et al.
1997).
Although the mechanisms are not fully understood, a tree is killed as a result of the simultaneous activities and interactions of both organisms, rather than the successive actions of beetles and pathogen (Nebeker and Hodges 1993, Paine et al. 1997). On the other hand, Six and Wingfield (2011) suggest that fungal phytopathogenicity has a more important role for the fungi, rather than supporting the bark beetles in tree killing. It may be a factor helping the fungi to survive within a living tree.
DEFENCE STRATEGIES OF NORWAY SPRUCE BARK
Due to the variety of pests the conifers evolve complex anatomical and chemical defences against pathogen attack, which may deter beetle invasion, impede fungal growth, and seal entrance wounds (Rohde et al. 1996, Bonello et al. 2001, Baier et al. 2002, Wermelinger 2004, Franceschi et al. 2005). Bark anatomy and the physiological condition of a potential host tree are crucial for the success of an attack/ infection. Until now, three types of tree's defence strategies have been described: constitutive, induced defences and systemic acquired resistance (Franceschi et al. 2005). Defence mechanisms are based on the bark anatomy and
March April	May	June July August		September
Spring generation Summer generation				
		■		
mm m		¡	m	
¡	ip ^ m		p ^ m>	
Fig.1: Generalised life-cycle of the spruce bark beetle (I. typographus), which normally has two generations per year - spring and summer generations. Bark beetles construct galleries, lay eggs and at the same time inoculate fungi into the bark phloem of woody plants(Six and Wingfield 2011).
10
Antioxidant defences of Norway spruce bark against bark beetles and its associated blue-stain fungus
its physiological condition. The main area of defence mechanisms is secondary phloem, which represents both physical and chemical protections (Hudgins and Franceschi 2004).
Constitutive and induced defence
An effective defence is provided by a multi-stage system, which runs as a series of different resistance mechanisms. Conifer trunk includes constitutive and inducible defences, which have attracted much attention over recent years. Most of the new knowledge has been obtained on resin-producing and resin-storing structures. Furthermore, quantitative and qualitative changes in the local metabolism have been studied around the entrance hole. These investigations have focused on phenolics, terpenoids and protein-based chemical defences (Brignolas et al. 1998, Evensen et al. 2000, Nagy et al. 2004, Wermelinger 2004, Franceschi et al. 2005, Erbilgin et al. 2006, Zeneli et al. 2006). Without prior attack the bark implements mechanical and chemical constitutive passive defence mechanisms. It involves initiation of a wound periderm, and based on bark anatomy also formation of cells with phenols, resin ducts, concentric layers of polyphenolic parenchyma (PP) cells, sclerenchyma and calcium oxalate crystals (Hudgins and Franceschi 2004, Franceschi et al. 2005, Hudings et al. 2004, Krekling et al. 2004).
An invasion activates inducible defence systems, which include secondary resin production, synthesis of new phenolics, and protein-based chemical defences. The induced defence detects attacks and closes the initial wounds by the formation of resin ducts, which contribute to increased synthesis of terpenes and resin flow. The induced defence is more effective and represents a more sustainable method of defence, as is provided by qualitative and quantitative changes in the chemical compositions of the trees' metabolisms. Furthermore, induced defence increases the formations of new PP cells and phenolic compounds, which are toxic for invasive organisms (Hudgins and Franceschi 2004, Franceschi et al. 2005, Schmidt et al. 2005, Witzel and Martin 2008). Pathogens use mechanical force or release cell wall degrading enzymes to break down this barrier. At the cell wall, they also trigger pathogen-associated molecular patterns (PAMPs) either inadvertently or as a consequence of plant degradative enzymes (e.g. the release of chitin oligomers by plant chitinases). Plants, in turn, appear to sense these PAMPs and damage to their cell walls and activate a variety of defences, including the production of reactive oxygen species (ROS), the production and export of anti-microbial compounds and fortification of their cell walls. PAMPs also activate a localised and systemic acquired resistance (LAR and SAR), initiating the synthesis of pathogenesis-related proteins (PR proteins), installation of lignin and accumulation of phenols and toxins (Hematy and et al. 2009). In addition, sensing PAMPs may activate intracellular defences such as the salicylic acid pathway, perhaps priming the plant for the next stage of warfare.
Collectively, constitutive and inducible compounds may deter beetle invasion, impede fungal growth, and seal entrance wounds (Bonello et al. 2001, Nagy et al. 2004, Wermelinger
2004, Franceschi et al. 2005, Zeneli et al. 2006, Rodrigues et al. 2009).
SAR - systemic acquired resistance
The last and very important defence strategy is composed of a systemic acquired resistance (SAR), which becomes affective during successful attack and ensures faster and easier responses to attacks in the future (Evensen et al. 2000, Percival 2001, Nagy et al. 2004, Franceschi et al. 2005, Bonello et al. 2006, Witzel and Martin 2008). This mechanism gradually develops throughout the plant during pest colonisation, and provides a systemic change in the whole tree metabolism. This leads to the production of fewer carbohydrates but more proteins, which are needed for defence (Viiri et al. 2001, Wermelinger 2004). The broad range of defence mechanisms, which contribute to the appearance of SAR, include antioxidants (ascorbic acid, cysteine, glutathione, total phenols) and antioxidant enzymes (Foyer and Rennenberg 2000, Riedle-Bauer 2000, Urbanek Krajnc 2004, 2009), which are generally linked to the actions of ROS (Hayat et al. 2009). Although anti-oxidative defence systems are often used as stress indicators for the diagnosis of disturbances in forest trees (Foyer and Rennenberg 2000, Riedle-Bauer 2000, Tausz et al. 2003, 2004, Grill et al. 2001, Tegischer et al. 2002), to date only a few investigations have dealt with anti-oxidative system of attacked Norway spruce (Urbanek Krajnc 2009, Urbanek Krajnc et al. 2014).
While several studies have demonstrated that the induction of phenolics occurs on a local scale (Evensen et al. 2000, Franceschi et al. 2000, 2005), several recent studies have also found evidence of the systemic induction of soluble low molecular weight phenolics (Bonello et al. 2001, 2006, Wallis et al. 2008). A SAR hypothesis postulated by Bonello et al. (2006) illustrated the interplay between SAR and induced susceptibility in trees against microbes and herbivores. The authors postulated that the time course of pathogen infection may have a bell-shaped effect on the strength of SAR that is similar to the spatial variations of constitutive secondary metabolites generated by resource availability. In the earliest stages of pathogen infection, SAR responses are predicted to rapidly and systemically increase concentrations of compounds involved in defence against pathogens and insects. However, if the pathogen is able to grow despite the deployment of localised defence responses, the infection will progress, and the tree will become increasingly stressed by the resulting resource limitations (e.g. reduction of sap flow, limited nutrient and water absorption, decreased carbon assimilation and growth). Consequently, a degradation of molecules involved in SAR would follow.
SAR has been actively studied in herbaceous plant species, and, over recent years, in woody plant species (Bonello et al. 2006, Eyles e tal. 2009), and is fast emerging as an intriguing, eco-friendly concept for enhancing tree resistance. However, before applying SAR becomes possible, there is a need to increase our knowledge of the mechanisms of defence in forest trees. Over recent years, research has focused on studying the impact of exogenous methyl jasmonate (MJ) and SA treatment on the inductions of certain metabolites
11
Antioxidant defences of Norway spruce bark against bark beetles and its associated blue-stain fungus
which are involved in SAR. Many authors (Franceschi et al. 2002, Hudings and Franceschi 2004, Hudings et al. 2004, Schmidt et al. 2005, Erbilgin et al. 2006) have examined the effect of MJ treatment in combination with the induced resistance of Norway spruce, where the positive impacts have been observed of MJ treatment and bark beetle attacks or Ceratocystis polonica infection. They reported that MJ treatment stimulated the synthesis and accumulation of terpenes (Kozlowski et al. 1999, Franceschi et al. 2002, Martin et al. 2002, 2003, Faldt et al. 2003, Hudgins and Franceschi 2004, Hudgins et al. 2004, Miller et al. 2005, Erbilgin et al. 2006, Zeneli et al. 2006).
In our previous field experiment (Urbanek Krajnc et al. 2011), 100 mM SA was applied to the bark sections of Norway spruce prior to being attacked by bark beetles, in order to study interactions with antioxidants and their significance for mediating stress-tolerance under natural conditions. SA-treatments significantly elevated the total SA levels over the whole sampling period. Total glutathione (tGSH) and total cysteine (tCys) increased, respectively, two weeks after treatment, in comparison with controls. One month after pheromone dispensers were placed on trees, an intensification of the ascorbate-glutathione system occurred within moderately-affected bark but to a greater extent after SA-treatment. Total SA levels within SA-treated moderately-affected trees remained at the control level until June. In contrast, strong attack was characterised by a successive increase in total SA and degradation of tGSH as well as total phenolics (tPH), a moderate increase in total ascorbate (tASC) and an oxidation of the ascorbate-glutathione pool within non-treated bark.
Furthermore, at the end of the three month experiment, the SA-treated bark had less entrance holes, and exhibited fewer and shorter maternal galleries than the control-bark. From this perspective, exogenous SA was successfully implicated as an activator of SAR in Norway spruce, providing tolerance against the complex interactive effects of bark beetle attack and environmental factors (Urbanek Krajnc et al. 2011).
OXIDATIVE STRESS AND ANTIOXIDANT RESPONSE OF CONIFER METABOLISMS
Antioxidants play an important role in chemical defence as they prevent the oxidations of other substances such as inactivate oxygen compounds and inhibit the formations of free radicals (Brodnjak Voncina 2006). Antioxidants are classified into three groups: real antioxidants that bind free radicals; reducing agents, while the third group consists of antioxidant synergists (Kugler 2011).
Both biotic and abiotic stress factors trigger plant metabolism changes and mostly react with the reactive oxygen species (ROS; H2O2). Oxidative stress reflects an imbalance between the systemic manifestations of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal redox states of cells can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell.
Antioxidants can slow down or even totally stop the oxidations of these molecules by hunting the free radicals and by removing oxidative damaged biomolecules (Kugler 2011, Veberic 2010, Harris and Hansen 2012). The antioxidants do not accumulate in the cytoplasm but in the vacuoles and other organelles such as plastids and mitochondria, where they are urgently needed as an effective "redox shuttle" (Kreft et al. 2000).
Phenolics
Among antioxidants phenolics represent a more important component of the inducible defence strategy regarding conifer bark. They range from simple, low molecular weight, single aromatic-ringed compounds to large and complex tannins and derived at polyphenols that provide different chemical and structural defence strategies.
In regard to the synthesis and accumulation of phenolic compounds, the barks of all conifer families have polyphenolic parenchyma cells (Krekling et al. 2000, Franceschi et al. 2000, 2005, Schmidt et al. 2005). Within their vacuoles, polyphenolic parenchyma cells contain variable amounts of phenolic bodies that are thought to serve as antifeedant and antifungal agents (Beckman 2000). Polyphenolic parenchyma cells are also major sites for storing starch and lipids (Krekling et al. 2000). In this capacity, they can be seen as targets for beetles and fungi, and constitutive phenolics can be hypothesised to protect the cells themselves, as well as prevent fungal penetration towards the cambial zone. In any case, the multiple layers of polyphenolic parenchyma cells providing physical and chemical resistance to penetration of the bark (Franceschi et al. 2000, 2005, Schmidt et al. 2005).
In our previous study on Norway spruce, the increase in total phenolics' concentrations two weeks after a bark beetle attack was recognized as an immediate inducible response to the attack (Urbanek Krajnc 2009, Urbanek Krajnc et al. 2011). Additionally, phenolics accumulated, to a higher extent, in the control tissue, which was explained as a wound reaction induced by mechanical injury (Christiansen et al. 1999, Franceschi et al. 2005, Ralph et al. 2006, Urbanek Krajnc 2009). The increase in total phenolics during the initial bark beetle colonisation was accompanied by the degradations of total ascorbic acid and tGSH, as well as oxidation of the ascorbate-glutathione pool. These initial events pointed out that the antioxidant defence is a multi-tier system with a spatial and temporal component. The spatial component is determined by the positions of PP cells' concentric rings from the periderm surface to the cambial zone (Franceschi et al. 2005) and the temporal component consisted of seasonal, continuous and enhanced production of phenolics in the attacked tissue. However, at later sampling dates, the trees with moderate attack were characterised by increased and steady-states of the tPH concentration, whereas the strong attacks were shown by drastically decreased concentrations of tPH (Urbanek Krajnc 2009). As the PP cells are the primary sites of phenolic biosynthesis within the secondary phloem (Franceschi et al. 2000, 2002, 2005), an assumption can be made that the synthesis of phenolics is lacking when the phloem is damaged, by the establishment of a complete
12
Antioxidant defences of Norway spruce bark against bark beetles and its associated blue-stain fungus
breeding system.
The more important phenolics in the Norway spruce phloem are stilbenes, flavonoids, as well as tannins (e.g. Lieutier et al. 2003, Schmidt et al. 2005, Witzell and Martin 2008, Hammerbacher et al. 2011). In general, phenolics fulfil different defensive functions. Most of the phenolics contribute to resistance indirectly. For instance, some low molecular weight phenolic compounds may function as precursors for other defensive compounds (e.g. lignin; Bonello and Blodgett 2003), or they may confer resistance as a group, rather than as individual compounds (Wallis et al. 2008). Catechin is a building block of condensed tannins for which antimicrobial activity via protein precipitation and iron depletion has been suggested (Witzell and Martin 2008).
Significant quantities of stilbene in pine bark were found years ago (Sjostrom 1993), while now it is known that pine bark extract contains several types of stilbene (the more important being astringin and isorapontin) and flavonoids. Changes in the concentrations of stilbenes and flavonoids in Norway spruce in response to injury or fungal infection are considered to be an active defence response (Brignolas et al. 1995, 1998, Viiri et al. 2001, Havsteen 2002, Schmidt et al. 2005, Witzell and Martin 2008, Li et al. 2012, Hammerbacher et al. 2011, 2013). Stilbenes are known to inhibit fungal growth by interfering with microtubule assembly (Woods et al. 1995, Adrian et al. 1997), disrupting plasma membranes and uncoupling electron transport within fungal spores and germ tubes (Pont and Pezet 2008, Adrian and Jeandet 2012). Stilbenes have also been shown to protect plants against oxidative stress (He et al. 2008), to deter herbivores (Torres et al. 2003), and to inhibit the growth of competing plants (Fiorentino et al. 2008).
Specific phenolic compounds in spruce inner bark against Ceratocystis polonica infection were previously interpreted by Urbanek Krajnc et al. (2014). Increases in tPH, catechin, astringin, isorhapontin and taxifolin were monitored. By analysing each single phenolic compound, on later sampling dates, it could be observed that within certain time shifts the phenolics followed the eco-physiological concept and basically fit with the temporal sequence of changes in tPH concentrations after the moderate bark beetle attack, previously reported on the spruce/bark beetle pathosystem (Urbanek Krajnc 2009, Urbanek Krajnc et al. 2011). The initial decline in catechin was followed by an increase in catechin concentration until September, when taxifolin accumulated within the infected bark at significantly higher levels until June, and later the concentrations dropped toward the control levels. On the other hand, a slight accumulation of astringin observed in response to fungi infection, when the concentration at later sampling dates dropped. The same was for isorhapontin concentration, where a dramatic initial increase was observed, and later it simply diminished (Urbanek Krajnc et al. 2014).
Ascorbate-glutathione cycle
Besides phenolics, ascorbate-glutathione system plays an important role in plant metabolism and defence. The ascorbate-glutathione cycle is considered to be the main pathway for ROS removal, and both ascorbate and glutathione
are recognised as the heart of the redox hub within the cell (Foyer and Noctor 2012).
Amongst antioxidants, glutathione is a low molecular sulphur metabolite, which plays multiple roles within tree-environment interactions and defences (Grill et al. 2001, Tausz et al. 2004, Zhao et al. 2008, Noctor 2006, 2012). It functions as a reductant in the enzymatic detoxification of ROS within the glutathione-ascorbate cycle and as a thiol buffer in the protection of proteins via direct reaction with ROS or by the formation of mixed disulphides (Zhao et al. 2008). In this role it has been suggested as a general redox sensor and signalling agent in plant cells (Meyer and Hell 2005, Cameron and Pakrasi 2010, Noctor et al. 1998, 2012).
Owing to its redox-active thiol group, GSH has often been considered as playing an important role in plant defence against oxidative stress (Grant et al. 1996). Trees under stress seem to generally require and synthetise higher concentrations of glutathione (Gullner and Komives 2001, Tausz et al. 2003, 2004). Glutathione synthesis depends on
NADPH
SOD ..................................T
-[Ca2+]c elevation
t
Defense-related genes Other responses?
Fig. 2: A model for the mechanism of SA action within the generation of active oxygen species, which induces the expression of the defence-related genes via an increase in [Ca2+] . SA and a trace of HO are used
L	Jc	2 2
for the SA-generating peroxidase reaction. Then the resultant SA' reacts with O2 to produce O2-that triggers an increase in [Ca2+] . The increased [Ca2+] may induce further physiological responses including the induction of PR genes. Ascorbate reconverts SA' to SA yielding monodehydroascorbate. An excess of ascorbate may inhibit the SA-dependent generation of O2-. While the generation of O2- is going on, SA inhibits the decomposition of H2O2 by catalase and a Fenton-type reaction. SA also lowers the HO' level by blocking the Fenton reaction and by directly trapping HO'. Thus SA protects the cells from highly reactive HO', while producing the less reactive O2- and H2O2 through a peroxidase-catalysed reaction, as the intermediate signals.
13
Antioxidant defences of Norway spruce bark against bark beetles and its associated blue-stain fungus
the distribution and cycling of sulphur in trees. Glutathione biosynthesis is restricted to the cytosol and the plastids, but mitochondria, which lack the capacity for glutathione biosynthesis, have been described as major sites of glutathione accumulations (Zechmann et al. 2007).
In all cells where GSH is found, besides reduced tripeptide form, the oxidised form of GSH (glutathione disulphide: GSSG) is also present in small quantities (< 20 %). While glutathione reductase (GR) uses NADPH to reduce GSSG to GSH, various free radicals and oxidants are able to oxidise GSH to GSSG (Fig. 2).
Both GSH and GSSG are considered as markers of oxidative stress in the plant, by increasing the antioxidant pool and as actors in cell signalling. Based on the current understanding of redox status and the roles of ROS, thiols, oxidisers and cellular antioxidants, scientists have redesigned a definition of oxidative stress (Harris and Hansen 2012), now it is based on changes in translation of thiol proteins, important for the control of the redox potential. Thiol redox couples such as GSH / GSSG, cysteine / cystine (Cys / cySS) form independent centres within the cells, which are linked to changes in the redox potential.
Glutathione is central to the regeneration of ascorbate within the ascorbate-glutathione cycle (Tausz et al. 2003, 2004). Glutathione, as an antioxidant, together with ascorbate, removes ROS, including hydrogen peroxide, superoxide and hydroxide radicals, which generate in cells as a result of oxidative stress (Xiang et al. 2001, Alscher 2006). In addition to being the most abundant water-soluble antioxidant in plant cells (Smirnoff and Wheeler 2000), ascorbic acid (AA) is also required for the re-conversion of SA, as ascorbate is highly reactive against phenoxyl radicals generated by peroxidases during oxidative stress (Fig. 2) (Kawano and Muto 2000, Smirnoff and Wheeler 2000). Together with glutathione, it is present in oxidation-reduction processes within the ascorbate-glutathione cycle. Dehydroascorbate (DHA) formed when ascorbate is oxidised, must be reduced again in order to prevent decreases in the total ascorbate pool (Likar and Regvar 2003, Sustarsic 2012). At alkaline pH values, GSH rapidly reduces DHA to ascorbate in a non-enzymic reaction. In plant tissues this reaction is catalysed by dehydroascorbate reductase (DHAR) (Noctor et al. 1998). The ascorbate glutathione cycle is essential for the removal of ROS and connects the conversion of ascorbic acid and glutathione.
Ascorbic acid is due to the two hydroxyl groups being one of the stronger reductants in the cell and allows the detoxification of reactive oxygen species. In the cell metabolism AA operates in oxidation-reduction processes, in the ascorbate-glutathione chain, where it is reversibly oxidised to DHA. At this stage, the oxidised form of ascorbic acid is restored via glutathione, when the GSH is oxidised to GSSG, and at the same time increases the GSSG / GSH ratio and the activity of GR (Foyer and Noctor 2012).
Although the antioxidant response is relatively well-documented on the level of spruce needles (Tegischer et al. 2002, Tausz et al. 2004), there is little known about changes in the ascorbate-glutathione chain at the inner bark level during bark beetle and fungus infection. In two of our previous experiments, the roles of thiols and ascorbate in the spruce
inner bark were studied during the five month sampling period after the spruce inner bark was exposed to beetle attack and Ceratocystis polonica infection (Urbanek Krajnc 2009, Urbanek Krajnc et al. 2014).
The initial reaction of Norway spruce to Ceratocystis polonica infection was characterised by a significantly more oxidised glutathione pool which was accompanied by higher GR activity. At the same time, the tCys dropped slightly below the levels of the non-infected samples and the cysteine redox state shifted towards a more oxidised value. Similarly, in one of the previous experiments, 2 weeks after the exposures of spruce trees to bark beetles slight but insignificant decreases were measured for tCys as well as tGSH, which were accompanied by a slightly more oxidised glutathione redox state (Urbanek Krajnc 2009). Until July, the infected inner bark was characterised by a gradual increase in tGSH, which was accompanied by a significantly increasing GR activity. Furthermore, within the infected inner bark the tCys remained elevated over the whole sampling period. Based on a previous experiment Urbanek Krajnc et al. (2009, 2014), concluded that the antioxidant shift within the Ceratocystis polonica infected bark indicated a successful defence reaction, which was characterised by a higher accumulation of thiols and a more reduced redox state. In September, the glutathione system reached a steady-state, and the GR activity and tCys concentrations remained increased. Consequently, thiols accumulated in May were accompanied by a significant degradation of tASC within the Ceratocystis polonica infected inner bark until July, followed by a significant accumulation in September. However, when comparing the tASC contents in the Ceratocystis polonica infected samples, the concentrations increased continuously from the spring onwards to September, which reflected increased pressure on the ascorbate-glutathione cycle (Urbanek Krajnc 2009, 2014).
CONCLUSIONS
Our previous field experiments (Urbanek Krajnc 2009, Urbanek Krajnc et al. 2011, 2014) demonstrated that the sequence of changes in the endogenous levels of antioxidant molecules within the affected Norway spruce inner bark strengthened the general eco-physiological stress-response concept as suggested by Larcher (2003) and Tausz et al. (2004).
The time-course analysis of the individual molecules enabled us to establish a relationship between salicylic acid, phenolics and the ascorbate-glutathione system in response to the complex interactive effects of bark beetle attack, infection by blue-stain fungus, and environmental factors (Urbanek Krajnc 2009, Urbanek Krajnc et al. 2011, 2014). Based on the ascorbate-glutathione concept of Foyer and Noctor (2011), phenolics within the concentric layers of PP cells can be viewed as a wheel of the bark defence mechanism, driven by the ascorbate-glutathione system, as a central part or redox hub of the wheel that integrates metabolic information and environmental stimuli to tone defence responses against pathogen and pests. The presented article reviewes the current knowledge on physiological and biochemical defense
14
Antioxidant defences of Norway spruce bark against bark beetles and its associated blue-stain fungus
responses of Norway spruce defence against bark beetles and associated fungi and also allows predictions of antioxidative defence responses in other conifer pathosystems.
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Antioksidativni obrambni odziv lubja navadne smreke ob napadu podlubnikov in z njimi povezanimi glivami modrivkami
IZVLEČEK
Čeprav so podlubniki in z njimi povezane glive sestavni del gozdnih ekosistemov, sta osmerozobi smrekov lubadar (Ips typographus Linnaeus) in z njim povezana patogena gliva modrivka [Ceratocystis polonica (Siem.) C. Moreau], ena najbolj uničujočih škodljivcev navadne smreke [Picea abies (L.) H. Karsten]. Lubadarji navadno naseljujejo oslabela in podrta drevesa, v specifičnih pogojih pa lahko napadejo tudi vitalna. S poškodbami floema in kambija ter motnjo transpiracijskega toka v ksilemu, drevesu povzročijo številne fiziološke motnje. Iglavci imajo široko paleto obrambnih mehanizmov, ki temeljijo na anatomiji lubja in fiziološkem stanju drevesa. Njihova osnovna funkcija je zaščita hranilno in energetsko bogatega floemskega tkiva, meristematske aktivnosti vaskularnega kambija in transpiracijskega toka v sekundarnem ksilemu debla. Glavno področje obrambnih mehanizmov je namreč sekundarni floem, ki predstavlja fizično in kemično zaščito lubja pred škodljivci, saj vsebuje polifenolne parenhimatske celice, sklerenhim, kristale kalcijevega oksalata in shizogene smolne kanale. Obrambni sistem lubja iglavcev zajema tri vrste obrambnih strategij - konstitutivno in inducirano obrambo ter sistemsko pridobljeno odpornost. Konstitutivna in inducirana obramba zavirata kolonizacijo podlubnikov, rast gliv in zapirata ranitvena mesta. Med uspešnim napadom pa je za drevo ključnega pomena še vzpostavitev sistemsko pridobljene odpornosti (SAR). Le ta se postopoma širi po rastlini ter izzove sistemske spremembe v metabolizmu drevesa, ki se ohranjajo skozi daljše časovno obdobje. Med številnimi obrambnimi odzivi, povezanimi z aktivacijo SAR, sta pomembni sinteza antioksidantov in aktivacija antioksidativnih encimov, ki varujejo rastlino pred reaktivnimi kisikovimi spojinami (ROS). Prispevek zajema pregled dosedanjega znanja o strategijah antioksidativnega odziva lubja smreke pri napadu smrekovega lubadarja (Ips typographus) in z njim povezane patogene glive modrivke (Ceratocystis polonica).
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Agricultura 12: No 1-2:19-24 (2015)
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The accuracy of the germination rate of seeds based on image processing and artificial neural networks
Uroš ŠKRUBEJa, Črtomir ROZMANb and Denis STAJNKOb*
aLokovica 8a, SI-3325 Šoštanj, Slovenia bUniversity of Maribor, Faculty of Agriculture and Life Sciences, SI-2311 Hoče, Slovenia
ABSTRACT
This paper describes a computer vision system based on image processing and machine learning techniques which was implemented for automatic assessment of the tomato seed germination rate. The entire system was built using open source applications ImageJ, Weka and their public Java classes and linked by our specially developed code. After object detection, we applied artificial neural networks (ANN), which was able to correctly classify 95.44% of germinated seeds of tomato (Solanum lycopersicum L.).
Key words: image processing, artificial neural networks, seeds, tomato
INTRODUCTION
As one of the most important input in agriculture a quality seed is a basis for higher agricultural productivity and a key to economic growth.
A number of methods for seed quality evaluation and sorting have been developed so far, mainly based on the detection of various physical and chemical properties which correlate well with certain vigour and germination parameters (McDonald 1998).
Nowadays, seed testing is performed in accredited laboratories by trained human analysts. The tests are designed to evaluate the quality of the seed lot. Several tests are done. For instance, a germination test determines the maximum germination potential, or viability of the seed. The germination rate of a particular seed lot is a key indicator which shows the seed performances in the field and it is expressed as a percentage (for example a 90% germination rate means 90 out of 100 seeds are likely to germinate under proper growing conditions). This information is important for calculating optimal seeding rates and to determine whether a particular seed lot has the potential to produce a quality crop.
Since the manual counting is time-consuming and labour
intensive process, we are looking at ways we can improve the process efficiency. We have been examining ways of automating a task by means of computer vision systems, based on image processing and machine learning. This can provide an alternative to manual counting and inspection of seed samples.
Image analysis was introduced in the field of seed technology already by Howarth and Stanwood (1994) who have developed a colour image database to characterize the phenotypic variation of genetic resources. Image processing also provided precise results in the field of seed identification or classification (Uchigasaki et al. 2000, Granitto et al. 2002) and germination assessment (McDonald et al. 1998). Dell'Aquila et al. (2000) used image analysis to characterize the imbibition of white cabbage seeds, while Geneve and Kester (2001) evaluated seeding size after germination by computer-aided analysis of digital images from a scanner (Ducournau et al. 2004).
Urena et al. (2001) proposed a machine vision system which used automated data gathering process and a fuzzy logic-based system for automatic evaluation of germination quality.
Ducournau et al. (2004) presented a machine vision system
*Correspondence to: E-mail: denis.stajnko@um.si
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The accuracy of the germination rate of seeds based on image processing and artificial neural networks
designed to count the number of emergent radicle tips on seed lots under controlled lighting, temperature and hygrometric conditions. The automated acquisition system employed an algorithm that was able to count the germinated seeds and provided the mean germination time based on the difference between two consecutive pictures.
Modern computer vision mainly based on image processing procedures such as proprietary software MATLAB or other specialized expensive software. In our work a free image processing and analysis program named ImageJ was used, which is readily available, open source and public domain software developed at the National Institutes of Health (NIH), Bethesda, Maryland USA (Rasband 2012).
MATERIALS AND METHODS
Tomato seeds (Solanum lycopersicum L.) variety 'Marmande', were obtained from the seed company Semenarna Ljubljana d.d. Slovenia. Before the experiment, the uncovered seeds were stored for a month in an incubator at 4 C°, 50% relative humidity to equilibrate to an identical seed moisture condition. Then we randomly chose 700 seeds from 3 bags as the sample. Next, we placed a dark filter paper inside twenty-eight glass Petri dishes (90x98x18mm) and moistened each with 3ml distilled water. The dark filter paper was used to obtain optimal contrast between seed, radicle and filter paper. Twenty-five seeds were placed on top of the wet filter paper in each dish and spaced them evenly. We put covers on the dishes. The seeds were germinated under a controlled condition and maintained in the dark at 20 to 30 °C (±1 °C) and 75% relative humidity for seven days in a Jacobsen incubator. The seeds were illuminated for 8 hours in every 24 hour period. Light was provided by a cool white fluorescent source of 750 lux. Images were captured by
a Nikon D80 digital SLR camera with Sigma 18 - 200mm zoom lens. The camera was mounted on a stand with an easy vertical movement, which provided rigid stable support. The camera was set at a distance 450 mm. The images were obtained by 3872x2592 pixels, horizontal resolution 300 dpi, vertical resolution 300 dpi and a bit depth 24. We placed a warm white, 22 W fluorescent tube with a 210 mm diameter circular lamp with a rated voltage of 220 V around the Petri dish with a seeds sample. A light diffuser, a semi-spherical steel bowl of 270 mm diameter, covered the light bulb, prevented external influences and provided diffused light (Figure 1). All images were transferred from the digital camera to a personal computer PC (dual-core microprocessor Intel Pentium B950 2.10 GHz, 4 GB installed memory RAM) via universal serial bus (USB) cable.
Images processing
The ImageJ software was used for image processing and extracting features from original RGB images (Figure 2a). First, we cut off the frame from each RGB image to establish the correct region of interest (ROI) in the centre of each image by using the known radius, so the cropped image was received (Figure 2b).The cropping process reduced the size of the images so all the following manipulations were more efficient. In our study the original matrix of 3872x2592 pixels was reduced to 1854x1836 pixels. In the second step a Gaussian filter with sigma parameter ff set at 2 was used for smoothing the image. This filter used a convolution with a Gaussian function (Eq. 1) described by Rasband (2008):
Fig. 1: Proposed computer vision system
(1)
where X is the intensity of pixel, ¡x is a mean,a is the standard deviation, <j~ is a variance, tt=3.18 and e=2.718. The image was then transformed from RGB color space into an 8-bit grayscale image (Figure 2c) which was finally converted by a threshold operation into a binary image (Figure 2d).For thresholding limits were defined by several pre-testing, so the lower limit was set to 85 and the upper limit to 255 grayscale intensity.
The minimum particle size was set to 1500 pixels and the maximum to 14.000 pixels. With this we additionally avoided the smaller and the bigger areas that could not be accounted as seeds. Finally, presented seeds were separated from the background and automatically labelled with the integer. The external perimeter of the seed was traced in yellow (Figure 2e).
The resulting set of extracted features together with statistics and measurements was saved in a separately table for each Petri dish, where each row represents a single seed and each column a single parameter of particular seed. Feature extraction was automated by using ImageJ's macro language facility, which enables any of ImageJ's GUI features to be invoked programmatically.
Simultaneously to image processing an expert technician
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The accuracy of the germination rate of seeds based on image processing and artificial neural networks
Fig. 2: Image processing. (a) Original image in RGB colour space. (b) Cropped image. (c) 8-bit grayscale image. (d) Binary image after thresholding. (e) Labelled seeds with numbers and yellow line. The next step in the processing was to extract the features from the image containing labelled seeds by Analyze Particles ImageJ command. The description of all measured and used image analysis parameters is reported in Table 1.
examined 28 images of each series of 25 seeds and provided a nominal class (germinated or not germinated) for each of the 700 seeds. Those results were also added to the previous mentioned tables.
Machine learning
On cethe feature vectors had been ge ne7 ated and exported as csv (comma-separated values) formatted file, we used machine learning software WEKA (Waikato Environment for Knowledge Analysis) to perform further analysis. WEKA is a collection of machine learning algorithms for data mining tasks and contains tools for data pre-processing, classification, regression, clustering, association rules, and visualization. The algorithms can either be applied directly to a dataset or called from Java code. WEKA was developed at the University of Waikato, New Zealand for the purpose of identifying information from raw data obtained from agricultural domains and due to the usability and openness extended also to other fields. It is written in Java and well connected with an ImageJ. The combination of these two tools was first time described by Mayo et al. (2007) for classifying the moths by species after feature vectors were extracted from each of the moth images.
Table 1: Parameters measured by ImageJ and ranked by WEKA
WEKA was first used for future selecting and ranking. For evaluator we used InfoGAinAttributeEvaluator which estimated the worth of an attribute by measuring the information gain with the respect to the class and Ranker which graded attributes by their individual evaluations. The biggest impact on classification had a parameter Perimeter which is the length of the outside boundary of the selection and lowest Mean which is the sum of the grey values of all the pixels in the selection divided by the number of pixels (Table 1).
For learning classifier models, the data were separated into 10 sets, each consisting of 70 instances. After that, the training was performed by using 9 of these sets, and testing was performed on the one remaining set (630 seeds as training and 70 seeds for testing for each run). This was repeated 10 times for each model.
Classification
For classification artificial neural networks (ANN) multilayer perceptron architecture (MLP) was implemented and compared with manual counting. For training we used a back propagation algorithm. The value of learning rate was set up
Parameter	Description
Perim	Perimeter is the length of the outside boundary of the selection.
Kurtosis	Kurtosis is the degree of peakedness of a distribution.
Max	The maximum grey values within the selection.
Skewness	Skewness is a measure of the degree of asymmetry of a distribution.
StdDev	Standard deviation of the grey values used to generate the mean grey value.
Major	Major is the primary axis of the best fitting ellipse.
Area	Area of selection in square pixels
Mode	Modal gray value is a most frequently occurring gray value within the selection. Corresponds to the highest peak in the histogram.
Median	The median value of the pixels in the selection.
Minor	Minor is the secondary axis of the best fitting ellipse.
Mean	Average grey value within the selection. It is the sum of the grey values of all the pixels in the selection divided by the number of pixels.
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The accuracy of the germination rate of seeds based on image processing and artificial neural networks
at 0.3 and momentum rate at 0.2. The number of neurons in input and output layers was set to 11 and 2 respectively since the number of features was 11 and the number of possible classes was 2. In the next step several combinations of hidden layers and different number of hidden neurons were tested, so the training time varied from 100 to 2000 epochs.
Evaluation measures for classifier performance
To objectively evaluate the performance of ANN, we used a classification accuracy, precision, recall and the F-measure, which were derived from confusion matrix.
Classification accuracy refers to the percentage of correct predictions made by the ANN model when compared with the manual evaluation of the 25 test data. It is calculated as the number of correctly classified instances divided by the total number of instances:
accuracy=(TP+TN)/(TP+TN+FP+FN)*100% (2)
where TP refers to true positive, TN refers to true negative, FP refers to false positive and FN refers to false negative. Then TP+TN+FP+FN is the total number of instances in the testing set and TP+TN is the number of correctly classified instances (Witten and Frank 2005).
In our case TP represented actually germinated seeds which were also predicted as germinated. TN represented actually not germinated seeds which were also predicted as not germinated. FP were actually not germinated seeds predicted as germinated and FN were germinated seeds predicted as not germinated.
Precision is the proportion of predicted positive instances which are actual positive among all those which were classified as positive. It is calculated as follows:
p=TP/(TP+FP)
(3)
where TP refer to true positive and FP refer to false positive. A FP occurs when the class is incorrectly predicted as positive
Fig. 3: ANN model with 11-4-2-2 topology
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The accuracy of the germination rate of seeds based on image processing and artificial neural networks
Table 2: Measures for classifier performance
model	Accuracy			Precision			Recall			F-measure		
	Mean	Std. dev	Sig.	Mean	Std. dev.	Sig.	Mean	Std. dev.	Sig.	Mean	Std. dev.	Sig.
ANN	95.44	2.14		0.9722	0.0233		0.9652	0.0218		0.9684	0.0148	
when it is actually negative (Witten and Frank 2005).
Recall is defined as the ratio of the true positive to the sum of the TP and FN. Recall is also known as sensitivity in some fields. It is calculated as follows:
recall=TP/(TP+FN)	(4)
FN occurs when the class is predicted as negative when is actually positive (Baeza and Riberio 1999, Witten and Frank 2005).
F-measure is defined as the harmonic mean of precision and recall. It is calculated as follows:
F-measure=(2xPrecisionxRecall)/(Precision+Recall) (5)
It has a high value when both precision and recall have high values, and it is seen as way of finding the best compromise between these two measures (Baeza and Riberio 1999).
RESULTS AND DISCUSSION
The best accuracy of 95.44% (Table 2) was obtained with 2 hidden layers that contained 4 and 2 hidden neurons, respectively, and a training time set at 500. The algorithm realized a 97% TP rate, with a 3% FN rate and a 93% TN rate with a 7% FP rate. ANN achieved the precision (0.9722), which is also very high. The ANN reached a recall (0.9652) and F-measure (0.9684), respectively.
The main advantage of our procedure over the study of (Dell'Aquila et al. 2000), who detected the germanised seeds by comparing the difference of seed XY position or area size of seeds on two consecutive images (before and after germination), was that we used eleven features derived from a single picture instead, to assess the germination rate. The single acquisition is also favourable, because it decreased a possibility of fungal contamination caused by several opening of the Jacobsen incubator.
Additional significant difference between our study and the one of Jossen et al. (2010) is that in our case the image processing, feature extraction and classification were entirely automatic, without any manual interventions for transferring data between different software applications.
CONCLUSIONS
In the present study, a computer vision system based on
image processing and machine learning techniques was developed which was implemented for automatic assessment of the seed's germination rate. The entire system was built by using open source applications ImageJ, Weka and their public Java classes and linked together by our specially developed code, which made it non-expensive and acceptable for many laboratories. The results show that ANN accuracy was very high (95.44%). The prototype system has classified one sample of germinated seeds in 4s and has out performed trained analysts, thus it shows a great opportunity for exchanging the time consuming manual counting in the laboratories for estimating the seed quality.
REFERENCES
1.	Baeza R, Ribeiro B. Modern Information Retrieval. Addison-Wesley Longman Publishing Co., Boston, 1999: 544 p.
2.	Dell'Aquila A, van Eck JW, van der Heidjen GWAM. The application of image analysis in monitoring the imbibition process of white cabbage (Brassica oleracea L.) seeds. Seed Sci. Res. 2000;10:163-169.
3.	Ducournau S, Feutry A, Plainchault P, Revollon P, Vigouroux B, Wagner MH. An image acquisition system for automated monitoring of the germination rate of sunflower seeds. Comp. Electron. Agricult. 2004;44:189-202.
4.	Geneve RL, Kester ST. Evaluation of seedling size following germination using computer-aided analysis of digital images from flat-bed scanner. Hortscience 2001;36(6):1117-1120.
5.	Granitto PM, Navone HD, VerdesPF, Ceccato HA. Weed seeds identification by machine vision. Comp. Electron. Agricult. 2002;33:91-103.
6.	Howarth MS, Stanwood PC. Extracting 3-D information using 2-D images of seeds. Comp. Electron. Agricult. 1994;10:175-188.
7.	Jossen R, Kodde V, Willems L, Ligterink W, Van der Plas L, Hilhorst H. Germinator: a software package for high-throughput scoring and curve fitting of Arabidopsis seed germination. Plant J. 2010; 62(1):148-159.
8.	Mayo M. Watson AT. Automatic species identification of live moths. Knowledge-Based Systems, 2007;20:195-202.
9.	McDonald MB. Seed quality assessment. Seed Sci. Res. 1998;8:265-275.
10.	Rasband WS. Image. US National Institutes of Health, Bethesda, Maryland, USA, 2008.
11.	<http://rsb.info.nih.gov/ij/index.html>	Accessed August, 2012.
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The accuracy of the germination rate of seeds based on image processing and artificial neural networks
12.	Uchigasaki M, Serata K, Miyamoto S. An automated machine vision system for classification of seeds using color features. J. Agricult. Struct. 2000;30(4):325-332.
13.	Urena R, Ro driguez F in B erenguel M. A machine vision system for seeds germination quality evaluation using fuzzy logic. Comp. Electron. Agricult., 2001,32:1-20.
14. Witten, I., and Frank, E. (2005).Data Mining: Practical Machine Learning Toolsand Techniques (2nd Ed.). Morgan Kaufmann,
Natančnost določanja kalečih semen s pomočjo obdelave slik in nevronskih
mrež
IZVLEČEK
Članek opisuje sistem računalniškega vida, ki temelji na tehnikah obdelave slik in strojnega učenja, ki je bil izdelan za avtomatsko oceno stopnje kaljenja semen paradižnika. Celoten sistem je bil zgrajen s pomočjo odprtokodnih aplikacij ImageJ, Weka in njihovih javno dostopnih javanskih kod, ki smo jih povezali v lastno originalno razvito kodo. Po odkrivanju predmetov na RGB slikah, smo uporabili umetne nevronske mreže (ANN), ki so bile sposobne pravilno razvrstiti 95,44% nakaljenih semen paradižnika (Solanum lycopersicum L.).
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Agricultura 12: No 1-2: 25-31 (2015)
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Impact of various types of anti-hail nets on light exposure in orchards and quality parameters of apples- a rewiev
Marinka BRGLEZ SEVER, Stanislav TOJNKO, Tatjana UNUK*
University of Maribor, Faculty of Agriculture and Life Sciences, Pivola 11, Hoce
ABSTRACT
The new Common Agricultural Policy (CAP) has been designed to shrink funds that Hungarian agricultural reform will ineNowadays, anti-hail nets are a part of basic equipment in a modern apple orchards. They decrease the risks of apple production and thus allow regular and quality apple harvest. Colours of nets differently obstruct the passing of light through the net, which directly affects some quality parameters of the yield, especially the fruit skin colour. The article includes a brief overview of studies associated with the impact of light exposure under different types of anti-hail nets on quality of apple fruits, as well as investigations which deal with adaptation of various technological measures, such as the use of reflective ground foil. A part of data from the existing literature explains the effect of anti-hail net usage on forming a microclimate under the net, and consequently, its effect on the development and dynamics of diseases and pests.
Key words: Anti-hail net, light, microclimate, apples, yield quality
INTRODUCTION
The benefits of anti-hail net usage are reducing the alternate bearing incidence due to protection of fruit trees, reduction of sunburn in hot years, protection against birds and insects, lesser impact of strong wind and continuous market supply of high quality fruits. Anti-hail nets point to the importance of consistently carrying out the technical support measures with the intention of balancing the growing conditions, mostly the light exposure of the fruit in the tree crown. (Germšek 2010, Germšek and Unuk 2014). As the distinct advantages of anti-hail net usage, Klein (2000) states a smaller day and night temperature variations under the net, a stronger and more uniform growth, and a partial protection against frost and of heavy rain.
Another benefit of using the anti-hail nets, as seen in practice, is also the possibility of combining it with the plant support systems. Due to the elimination of production risks, which in some regions poses a distinct problem because of the hail, the placement of anti-hail nets allows for a quicker return of the invested assets in aorchard establishment, earlier
and more regular yields of high quality, and thus a higher economy of apple production (Germsek in Unuk 2014).
Anti-hail net light transmittance
Light has a crucial role when it comes to the intensity of the photosynthesis, differentiation of floral buds, and consequently also affects the quality of fruit. It is also a key factor affecting colour, taste and resilience of fruits (Jazbec and Hanzak 1982, Holzwarth 2008).
Photosynthetically active radiation (PAR), which is needed for photosynthesis, has the wavelength between 400 and 700 nm. Approximately from 85 to 95% PAR - radiation is absorbed by a leaf, the rest is either reflected or penetrates the leaf (Stampar et al. 2002). The level of light is decreased for approximately 7% (PAR) - 20% (UV) with a white net (which allows for the maximum light exposure under a net): 11% (PAR) -28% (UV)with a red-white net, 12% (PAR) -23% (UV) with a green-white net, 13% (PAR) with a light grey net, 15% (PAR) - 26% (UV) with a black and green, 16%
*Correspondence to: E-mail: tatjana.unuk@um.si
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Impact of various types of anti-hail nets on light exposure in orchards and quality parameters of apples- a rewiev
(PAR) - 23% (UV) with a black net and 18% (PAR) - 29% (UV) with a red net, measured 50 centimetres below the net. The level of UV light is decreased by 29% with a black net (Blanke 2007). With a grey net the level is decreased by 13% (PAR), whereas with a black net by 18% (PAR) (Blanke 2007, cited.).
Larcher (2003) states that the leaves exposed to the sunlight, compared to those in the shade, have a higher level of net photosynthesis. The leaves on the trees protected by anti-hail net could behave similarly to the leaves in the shade, due to somehow decreased exposure to light. Widmer (2001) claims that swings in day temperature show thet weather (cloudiness, fog) can affect the light exposure to a much higher degree than the anti-hail net; he states that the assimilation of leaves experiences a small decrease only on cloudy days, while it remains unchanged on sunny days.
A part of UV rays is transmitted through the nets as a visible light, therefore they do not represent additional protection against UV rays with the wavelength above 300 nm. Trees and fruits under such nets are, during their growth, exposed to the same level of UV - A and partially UV - B radiation as well as the level of visible light. The effect of UV radiation on plants depends on strength of the visible light and is even stronger when the light is weaker, for example in dapple shade, in cloudy weather and under the net. For fruit farming, nets with higher level of light transmittance in regard to durability of materials and price are recommended. It's not yet entirely clear which part of the radiation they reflect (Nobel 1991, cit. in Germšek 2010).
Different types of anti-hail nets obstruct the passing of the light differently and have different life expectancies. The thinner the net and the bigger the meshes, the higher are the swings in transmittance levels (Blanke 2007). Grey nets represent the biggest share of the standard production. They consist of crystal fibres with added black fibres for better durability. Black anti-hail nets, in comparison to white ones, have longer life expectancy, lasting from 15 to 20 years, and blend into the colour composition of the landscape more easily, but decrease the light transmittance by 20% (Zadravec 2002, Germšek and Unuk 2014). White nets transmit more light (86%), but at the same price come with less than a half of the life expectancy of a black net (Dobaja 2005); white net's life expectancy is between 7 and 9 years. Its weakness is also in its noticeability in nature, compared to the black net, which led to a compromise in net colours - grey and green nets. Due to dirt accumulation over the years, the difference in light transmittance between the types of nets decreases with time (Dobaja 2005). Light transmittance in sunny weather is lower with white and black net compared to the uncovered part. With the black net it is decreased by 25% and with the white one by 12%. With red-white nets the transmittance is decreased by 14%, with red-black ones by 18% and with green-black ones by 23% (Blanke and Salomaklin 2008). Under a grey net, shading reaches up to 12% (Zadravec 1998, as cited in Kuzma 2011). Iglesias and Alegre (2006) proved that the differences in light transmittance due to the colour of the net were smaller in cloudy weather.
Blanke (2007) states that in the ecological conditions of Southern Europe, for single-colour and two-colour varieties of apples, which achieve apropriate colouring, or
for varieties which are prone to sunburn, black nets are the most appropriate. White or crystal and grey nets are more appropriate for the apple orchardsin NW Europe, where the lack of sunlight is noted and where there is no risk of sunburn. Red nets seem inappropriate because of the higher level of shading and also because they cannot blend with the landscape.
Anti-hail nets are mostly made out of highly compressed polyethylene fibres, 228-356 ^m wide. One of special characteristics of black and black-green nets is that they have two black longitudinal threads, while transparent and red nets have two transparent or two red longitudinal threads. Transverse single threads in black, red and green nets are black. Nets also differ in thread density. The anti-hail net with double knitting lets through only the smallest possible grain of hail. It has a double longitudinal and a single transverse knitting (Blanke 2007).
Light exposure under the anti-hail net and substituting the lack of light with a reflective ground foil
The fruit skin (fruit exterior) colour represents one of the basic parameters which attract the consumers; in the eyes of a buyer, it represents direct connection to the internal quality of the fruits (Sadar et al 2013). This is the main reason why growers use numerous measures to improve the colouring of fruits. Because of the decrease in light transmittance in orchards under the anti-hail net, the skin colour is even a bigger challenge for the growers.
Blanke (2004) states that a white reflective foil laid out between the rows of trees, improves the colouring of fruits of the 'Jonagold Wilmuta' variety. This impact is even bigger when the foil is laid out early, meaning in July instead of August. The improved colouring of the fruits with the use of reflective foil is confirmed in combination with the white and black net. It was established that the improved colouring of the fruits is achieved under white and black nets, if the reflect foil is laid on the ground at least six weeks prior to the harvest. While studying the impact of the reflective foil, Jakopič et al. (2007) confirmed that the foil under the net could compensate the decrease of light transmittance even in the last month before harvest, and that covering the ground of the orchard with the reflective foil had a positive effect on the fruits' (variety 'Fuji') exposure to light.
In the ecological conditions of the Fruit Growing Centre in Maribor (NE Slovenia), the impact of the anti-hail net and the ground reflective foil on the size and quality of yield (variety 'Jonagold' and 'Elstar') was not documented. However, the positive impact of the reflective foil on the fruit colouring was confirmed. The impact was the highest in combination with the crystal net (91.7% on the variety 'Elstar'). The highest impact on fruit colour ofthe variety 'Elstar' was recorded with the use of reflective foil (91.6%), followed by the crystal net (91.7%), the black net (84.9%), control - without nets and reflective foil (84.1%), reflective foil in combination with the black net (80.8%) and reflective foil in combination with the crystal net (79.7%). In 2002, the highest impact on fruit
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Impact of various types of anti-hail nets on light exposure in orchards and quality parameters of apples- a rewiev
colour of the variety 'Elstar' was recorded with the use of crystal net (64.4%), followed by reflective foil in combination with the crystal net (58.3%), control (54.5%), reflective foil in combination with the black net (51%) and reflective foil (47.7%). Regarding the variety 'Jonagold', the highest impact on fruit colour in 2001 was recorded when using the reflective foil in combination with the crystal net (61.5%), followed by the crystal net (58.8%), the black net (58.5%) and reflective foil in combination with the black net (47.8%). In 2002, the highest impact on fruit colour of the variety 'Jonagold' was recorded when using the reflective foil in combination with the black net (78.1%), followed by reflective foil (59%), the crystal net (58.6%), the black net (41%), reflective foil in combination with the crystal net (34.1%) and control (21.7%) (Dobaja 2005).
The impact of anti-hail net on some quantity and quality parameters of fruits
The optimal light conditions in the tree crown improve the health status and colour of the fruits (anthocyanin synthesis) (Awad et al. 2001). Light is very important for the increase in the anthocyanin level (Macheix et al. 1990). The less light reaches the treetops, the smaller is the intensity of photosynthesis, which in the end results in the lesser fruit colouring (Ubi et al. 2006). Different authors (Vercammen 1999,Widmer 2001,Zadravec 2002, Germsek and Unuk 2014) prove that the quantity of yield under the protective net does not decrease; many of them even state the opposite. Zadravec et al. (2009) and Germsek (2008) proved that the net does not impair the bearing fruit - on the contrary, they noted a better differentiation of flower buds on the trees under the net (which might also be due to the uncovered trees being damaged by hail). Anti-hail net does not affect the trunk width, growth of the annual shoots, the number of nodes, or any other aspect of the exuberance of growth (Germsek 2008).
Widmer (2001), Blanke (2007) and Zadravec et al. (2009) established that the external quality of the fruit (diameter) under the net does not worsen. Germsek and Unuk (2014) state that the average weight of fruits is bigger under the black net, while Zadravec (2002), Stampar et al. (2002) and Iglesias and Alegre (2006) did not notice any affect on the average weight of the fruits.
Internal quality is also becoming more and more important for the consumer. Many authors have established that the internal quality of fruits somewhat changes when grown under an anti-hail net; fruit firmness and acid content increase in certain years, but the content of dry matter typically decreases (Widmer 2001, Steinbauer 2008, cit. in Zadravec et al. 2009). Zadravec (2002) establishes that there is more soluble dry matter in fruit grown outside the net (fruit with better colour contains more dry matter). Likewise, Germsek and Unuk (2014) establish that apple fruit (variety 'Gala Brookfiel' and 'Fuji Kiku 8') grown under the black anti-hail net has a lower content of dry matter (from 0.6 to 1 °Brix). Stampar et al. (2002), Iglesias and Alegre (2006), Germsek and Unuk (2014) claim that hardness of the fruit is not linked
to the anti-hail nets. Stampar et al. (2002) also establish at that cultivation under anti-hail nets does not influence the content of malic and fumaric acid, although the fruit grown under a black net contains more citric acid.
Zadravec (2002) and Germsek and Unuk (2014) noted that the starch decomposition in the fruit is slower under a net, which is a sign of slower ripening. More dry matter was measured in the fruit grown without a net (fruit with better colour contains more dry matter) and a lower starch index was established in the fruit grown under a net (Stampar et al. 2002, Zadravec 2002, Germsek and Unuk 2014). An anti-hail net causes minimal differences of individual sugars (2x more glucose and fructose than sucrose) (Zadravec 2002). Stampar et al. (2002) prove the differences in glucose content (higher under black) and sucrose (higher under white), with no differences in fructose and sorbitol.
Fruit grown under the net is less exposed to sunburn, despite unchanged maximal leaf temperatures on trees (Zadravec 1998, cit. in Kuzma 2011). A lower risk of sunburn under anti-hail nets, especially under black nets, and consequently higher quality of the fruit's skin is also established by Stampar et al. (2002), Dussi et al. (2005), Iglesias and Alegre (2006). Zadravec (2002) establishes that 12% of fruit was sunburnt on trees without a net, 6% on trees under a white net and 5.5% on trees under a black net. Therefore, a reduction of light exposure is very welcome in extreme circumstances. Concerning the presence of sunburn, Steiermark recommends the use of black nets, woven with two fibres. Their experience shows that there was no sunburn on trees planted under a black net, whereas 6.5% of fruit was sunburnt on trees grown without a net; when a reflective foil was used for better fruit colour 9% of the fruit was sunburnt, on organic trees, where lime sulphur was used to protect from diseases, 16% of fruit was sunburnt (Caf 2009, cit. in Germsek 2010).
Casierra-Posada and Ludders (2001), when studying the effect of summer pruning and mineral content of apple trees under anti-hail nets, have determined that anti-hail nets effect the content of minerals in sprouts and leaves. When pruned in June, the content of calcium and magnesium was higher in leaves. The fruit grown outside of a net had a higher content of calcium and potassium in comparison to the fruit grown under an anti-hail net. The fruit grown outside of a net has a higher calcium - potassium ratio in comparison to the fruit grown under a net. Growing fruit under a net decreases the ration between (K+Mg) / Ca.
Fruit skin colour under an anti-hail net
The basic colour of apples comes from chlorophyll pigments, most of which is chlorophyll a and b in a ratio 3 : 1. During ripening, the green colour turns to yellow, because of the forming of carotenoids (Hribar 1989). The ripening of red fruits leads to red colour in the fruit's skin. The decrease of green colour is connected to the decomposition of chlorophyll, while the red colour stems from the accumulation of anthocyanin (Macheix et al. 1990).The visual perception of colour is not only effected by the absolute concentrations of individual pigments in the fruit's skin but also by the proportions of the vacuoles and the distribution and size of
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Impact of various types of anti-hail nets on light exposure in orchards and quality parameters of apples- a rewiev
the cells in the apple's skin. The final percept of the colour is a consequence of a combination of all previously listed factors (Curry 1997).
It has been proven that a net can influence the chlorophyll synthesis in leaves (depending on variety, growth period and planting distance), as the average levels of photosynthesis pigments in leaves under a net are lower than outside of it. According to Horvat (2011), anti-hail nets also have an influence on the smaller decomposition of chlorophyll and formation of red pigments in fruits' epidermis, which has been confirmed by Germšek and Unuk (2014). Because of the small variability in the fruit grown under the net, compared to that grown without a net, Germšek and Unuk (2014) claim, that at the time of the technological ripeness the an anti-hail net (black) does not have a negative impact on fruit skin colour in cases where the study was conducted on the better coloured clones (like the variety 'Gala Brookfield').
Also according to Preiffer and Jankovič (2004), the net has a positive effect on the fruit's colour, because the colouration of the fruits is more simmilar. The fruit skin colour is mostly genetically determined and depends on weather conditions, special Low night temperatures during ripening have a positive effect on the accumulation of anthocyanins in apples. Ubi et al. (2006) claim that low temperatures cause a lower consumption of carbohydrates for breathing and increase photosynthesis, which ultimately affects the biosynthesis of anthocyanin. Night time temperatures between 3 and 11 °C are supposedly most suitable for coloration (Iglesias and Alegre 2006). Optimal daytime temperatures for anthocyanin accumulation are between 15 and 25 °C,whilst optimal night time temperatures are between 10 and 20 °C. Vidrih and Hribar (2002) claim that the fruit's colour is more intense when the differences between day and night time are bigger, and that coloration is optimal if cold nights (10-15 °C) are followed by warm (20-25 °C) and sunny days.
Horvat (2011) believes that the research of anthocyanin content shows that anti-hail nets effect and slow down the decomposition of chlorophyll and formation of red pigments in the fruit's epidermis. Germšek and Unuk (2014) point to an average 7 day (variety 'Gala') and an average 10 day (variety 'Fuji') delay in the decomposition of chlorophyll in the fruit's epidermis.
This effect was reduced at the time of optimal technological ripeness or was not perceived in favourable weather conditions at the time of ripening.
Concerning fruit colour, Jakopič et al.(2007) states that the variety 'Fuji' shows no differences in colour under a net or outside of it. Zadravec (2002) claims that the use of anti-hail nets causes an inferior fruit colour, which is most obvious when the black net is used. In another experiment, Zadravec et al. (2009) establishes no negative impact of anti-hail nets on fruit colour, just a slight reduction of the quantity of fruits with the most desired colour.The red skin colour is slightly worse in two-coloured varieties, while fully red varieties show no negative impact on the colour of the epidermis (Blanke 2007). Germšek and Unuk (2014) have made an effort to show that the position of the fruit (variety 'Gala Brookfield' and 'Fuji Kiku') has a stronger effect on the dynamics of colour development than the anti-hail net itself.
The impact of anti-hail nets on the microclimate of the orchard
In an experiment that lasted several years in the Fruit Growing Centre Maribor - Gačnik, the measurements of daily temperatures have shown that nets somehow reduce momentary temperature fluctuations caused by sun exposure changes. Similar results were found when measuring relative air humidity. A smoother curve in daily temperatures and relative air humidity can mean somehow better or less stressful conditions for a tree, which could explain why the reduction of light does not result in lower production capabilities of the orchard under an anti-hail net (Zadravec 2002).
The use of the net in an experiment in Spain has shown differences in temperature under the net and outside of it. The maximum temperatures under the net were 3 °C lower, but only on sunny days. On cloudy days, the differences were negligible. The decrease of maximum temperatures are also confirmed by Torggler (2002) (cit. in Zadravec et al. 2009) by, from 0.3 to 0.4 °C, Kuhrt et al. (2006) by 0,8 °C, and Solomakhin and Blanke (2008) by 0,2 °C, but only on sunny days. Zadravec (1998, cit. in Kuzma 2011) states that the somewhat smaller differences between maximum and minimum air temperatures could partly be the reason for delayed and lower fruit colouration.
Iglesias and Alegre (2006) also found a slight increase in relative air humidity under a net.They found a 2-6% increase of relative air humidity and, at the same time, a 11% decrease in evapotranspiration, which wasassociatedwith lower wind velocity. In comparison to measurements outside the net, an increase in relative air humidity from 0 to 8.9% was also determined under an anti-hail net by Torggler (2002, cit. in Zadravec et al. 2009).
A positive effect of anti-hail nets on fruit temperature has also been shown. Reduced fruit and leaf temperatures consequently mean less sunburn damage. A black net reduced the temperature of fruit by 4 °C, a white net by 2.5 °C. On cloudy days with less light, the differences were smaller. The differences between air and fruit temperatures were also smaller than on sunny days (Iglesias and Alegre 2006).
The anti-hail net also affects the quantity of rain that gets to the ground. The differences in the measurements of rain fall(less under an anti-hail net) are probably the consequence of the measuring instrument accuracy, and only with very small quantities of rain would it be possible that a part of the rain is "intercepted" by the net and evaporates from it - so the rainwould not actually fall on the plants and ground in the orchard under the net (Zadravec et al. 2009).
The duration of the leaf dampness after rain, which is (c. 1 hour) longer under an anti-hail net, could only change the course of the incubation period for the apple scab in very specific infection circumstances. It is believed that black anti-hail nets do not prolong leaf dampness significantly and therefore do not increase the danger of certain diseases (Zadravec et al. 2009).
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Impact of various types of anti-hail nets on light exposure in orchards and quality parameters of apples- a rewiev
The impact of anti-hail nets on disease and pest development and mobility
Reigne (1997) (cit. in Iglesias and Alegre 2006) argues that anti-hail nets do not have an effect on the occurrence of the codling moth. Demaria et al. (2006, cit. in Iglesias and Alegre 2006) and Zadravec et al. (2009) observed that there was a lower number of this pest under an anti-hail net and the damage to fruit was lower when compared to the part which was outside the net. Ferjanc (2009) believes that the net hinders the movement of codling moths, but claims that despite this there is no distinctive impact on the degree of fruit damage. Graf et al. (1999) have established that the net is an obstacle when the codling moths move in, but mostly does not hinder these moths moving out. In investigations associated with the effects of nets on mating of codling moths, there were no differences between the part of the orchard under the net and the part outside it, when the lure was placed on the height of 1.5 m, while the difference was visible when the lure was placed on the height of 3 m, as there were more fertilised females outside of the net (Tasin et al. 2008). Kuhrt et al. (2006) claim that individual stages of the codling moth development occur a few days later under the net because of lower temperatures. In accordance with thermoregulation in the phenology model, the stages appear four days later under the net, except for the first generation, where the occurrence is three days late. The development of the whole generation was one day shorter than outside the net. The delay of the occurrence of individual stages under the net, where thermoregulation was not integrated, was four days for all stages.
Reigne (1997, cit. in Iglesias and Alegre 2006) establishes that anti-hail nets do not influence the occurrence of plant lice, while Racsko et al. (2005) states the importance of temperature in the development of this pest. Because of lower wind speeds under the net, the migration conditions of plant lice are better, while the possibility of other pests is decreased. They also believe that delayed ripeness does not influence the development of larvae in the fruit. Ferjanc (2009) states that anti-hail nets with regular mesh size cannot physically prevent the plant lice from entering the orchard. He believes that the impact on the size of the plant lice population varies according to the type and manner of migration between hosts.
Graf et al. (1999) and Tasin et al. (2008) have established that more fruit was damaged from caterpillars in orchards outside of nets. Zadravec et al. (2009) claim that the plant lice population develops faster and better under an anti-hail net. Ferjanc (2009) believes that the red plant lice develops better outside the anti-hail net, but states that the impact on the occurrence of this pest differs depending on fruit varieties ('Braeburn', 'Gala' and 'Fuji').
Reigne (1997) (cit. in Iglesias and Alegre 2006), Zadravec et al. (2009), Ferjanc (2009) notethat anti-hail nets do not have an effect on diseases, such as, for example, apple scab and powdery mildew. Ruegg (1997) (cit. in Widmer 2001) also emphasizes that the leaves under the net, compared to those outside the net, do not stay damp long enough, which is why the possibility of apple scab infections is lower. Zadravec et al. (2009) state that the duration of the leaf dampness after
rain, which is (app. 1 hour) longer under an anti-hail net, could only change the course of the incubation period for apple scab in very specific infection circumstances.
CONCLUSION
Numerous researchers have demonstrated different experiences with anti-hail nets. It maybe reasonable to set experiments for each specific situation, where a wider selection of different types of anti-hail nets (with different colours and structures) would be integrated. In order to obtain reliable data, more experiments would have to be carried out in individual topics of great interest (the impact of anti-hail nets on the development and movement of pests, development of diseases, fruit quality, vegetative reactions of cultivated plants to conditions under anti-hail nets...), since the current research shows that some subjects and factors are poorly explored and that information on the effects of anti-hail nets is incomplete. In the future, it would beuse ful that experiments associated with anti-hail nets and their impact would include less explored topics (irrigation needs, tendencies of natural undeveloped fruit pruning under anti-hail nets...). More coherent findings and a more comprehensive understanding of these impacts in different researches would lead to more uniform conclusions which are urgently needed in practice.
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11.	Germšek B. Razvoj parametrov kakovosti jabolk sort 'Gala' in 'Fuji' kot posledica vpliva protitočne mreže in položaja plodov v krošnji [magistrsko delo]. Maribor: Fakulteta za kmetijstvo in biosistemske vede, Univerza v Mariboru, 2010,
12.	Germšek B, Unuk T. Kakovost jabolk sort 'Gala Brookfiel' in 'Fuji Kiku' pod in izven protitočne mreže. Acta agricult. Slov. 2014;103(1):137-144.
13.	Graf B, Höpli H, Rauscher S, Höhn H. Hagelnetze beeinflussen das Migrationsverhalten von Apfel - und Schalenwickler. Obst - Weinbau 1999;12:289-292.
14.	Holzwarth R. Hagelschutznetze: Moderne Technik schützt das Obst. Besseres Obst 2008;6:25.
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16.	Hribar J. Spremembe kemičnih in mehaničnih lastnosti jabolk sorte 'Jonagold' pri različnih pogojih skladiščenja [doktorska disertacija]. Ljubljana, BF, VTOZD za živilsko tehnologijo, 1989,
17.	Iglesias I, Alegre S. The effect o anti - hail nets on fruit protection, radiation, temperature, quality and profitability of 'Mondial Gala' apples. J. App. Horticult. 2006; 8(2):91-100.
18.	Jakopič J, Veberič R, Štampar F. The effect of reflective foil and hail nets on the lighting, color and anthocyanins of 'Fuji' apple. Sci. Horticult. 2007;115(1):40-46
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22.	Kührt U, Samietz J, Höhn H, Dorn S. Modelling the phenology of codling moth: Influence of habitat and thermoregulation. Agric. Ecosyst Environ. 2006;117:29-38.
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24.	Macheix J.-J, Fleuriet A, Billot J. Fruit phenolics. Boca Raton, CSR Press, 1990,
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Wiesel. V: Zbornik referatov 1. Slovenskega sadjarskega kongresa z mednarodno udeležbo. Krško, 24.- 26. marec. Ljubljana, 2004:249-253.
26.	Sadar N, Urbanek Krajnc A, Unuk T. 2013. Spectrophotometrically determined pigment contents of intact apple fruits and their relations with quality: a review. Zemdirbyste-Agriculture vol. 2013;100:105-111., No. 1 (2013)
27.	Steinbauer L. Treffen der Arbeitsgruppe »Obstbau unter Hagelnetzen. Haidegger Perspektiven 2008;3:6-7.
28.	Štampar F, Veberic R, Zadravec P, Hudina M, Usenik V, Solar A and Osterc G. Yield and fruit quality of apples cv. 'Jonagold' under hail protection nets. Gartenbauwissenschaft 2002;67(5):205-210.
29.	Tasin M, Demaria D, Ryne C, Cesano A, Galliano A, Anfora G, Loriatti C, Alma A. Effect of anti-hail nets on Cydia pomonella behaviour in apple orchards. Entomologia experimentalis et applicata 2008;129(1):32-36.
30.	Ubi BE, Honda C, Bessho H, Kondo S, Wada M, Kobayashi S, Moriguchi T. Expression analysis of anthocyanin biosynthetic genes in apple skin: Effect of UV - B and temperature. Plant Sci. 2006;170(3):571-578.
31.	Vercammen J. Eerste ervaringen met hagelneten - ten in Belgie. Fruitteelt - nieuws 1999;12:6-8.
32.	Vidrih R, Hribar J. Optimalni rok obiranja sadja. Brstika. Priloga tednika kmečki glas za sadjarje in vinogradnike. 2002;1,3:4-5.
33.	Zadravec P. Vpliv protitočne mreže na količino in kakovost pridelka pri jablani. Sodobno kmetijstvo. Kmečki glas 2002;35(3):108-111.
34.	Zadravec P, Donik B, Beber M, Unuk T, Tojnko S, Lešnik M, Germšek B, Ferjanc B. Odziv jablane in škodljivih organizmov na spremenjene razmere pod protitočnimi mrežami. Monografija, Sadjarski posvet, 2009:38-44.
35.	Widmer A. Beschattung unter weissen und grauen Hagelnetzen. Obst und Weinbau 2001a;133:581-583.
36.	Widmer A. Light intensity and fruit quality under hail protection nets. Acta Hort. 2001b;557:421-426.
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Impact of various types of anti-hail nets on light exposure in orchards and quality parameters of apples- a rewiev
Vpliv različnih tipov protitočnih mrež na prepustnost svetlobe in parametre kakovosti jabolk - pregledni članek
IZVLEČEK
Danes protitočne mreže predstavljajo osnovno opremo v nasadih jablan. Zmanjšujejo rizik pridelave in hkrati omogočajo redne pridelke jabolk visoke kakovosti. Različne barve mreže različno močno ovirajo prehod svetlobe, kar ima neposredni vliv na parametre kakovosti pridelka, posebej obarvanost. Članek vkjučuje kratki pregled objav, povezanih s prepustnostjo svetlobe svozi različne tipe protitočnih mrež ter posledično vpliv na kakovost plodov. Vključene so tudi vsebine raziskav, ki tečejo na temo prilagoditve posameznih tehnoloških ukrepov v nasadu, kot je to npr. uporaba reflektivne folije. Nekaj zbranih informacij, ki jih lahko najdemo v literaturi, pa pojasnjuje vpliv protitočnih mrež na vzpostavitev mikroklime v nasadu ter posledično vpliv na razvoj in dinamiko pojava posameznih bolezni in škodljivcev.
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Agricultura 12: No 1-2: 33-40 (2015)
DE
G
DE GRUYTER
OPEN
Sweet maize growth and yield response to organic and mineral fertilizers,
N rates and soil water regimes
Franc BAVEC1*, Martina BAVEC1, Silva GROBELNIK MLAKAR1, Milojka FEKONJA2
University of Maribor, Faculty of Agriculture and Life Sciences, Pivola 10, 2311 Hoče, Slovenia 2Development Agency Slovenske gorice, Trg osvoboditve 9, 2230 Lenart
ABSTRACT
Sweet maize is an underutilized vegetable in European temperate areas, and its consumption is increasing. For better understanding of cultivation practices, this pot experiment aimed to determine the effects of different water regimes and nitrogen (N) rates calculated from N target values. N rates of 0 (control), 0.6 and 2 g N pot-1 were applied as organic by-products pumpkin cake and pig manure digestate, and mineral fertilizers CAN 27 and ENTEC®26. Treatments of water supply were based on measured soil matric potentials of 2.8 pF (drought stress), 2.6 pF (optimal water) and 2.4 pF (overwatered). In comparison to mineral fertilizers, pumpkin cake proved to be equal in effectiveness in plant height (155.8 cm), cob (85.8 g), green (124.9 g) and leaf mass per plant (44.2 g), or even better in root (72.3 g) and broom mass per plant (3.0 g). Yield parameters, cob mass (70.1 g), its length (6.3 cm) and diameter (2.0 cm), as well as the residual mineral N (59 mg N kg-1) significantly increased at the highest N rate. Significantly lower values of the evaluated morphological parameters and photosynthetic rates (at brooming and harvesting) were associated with drought stress. The matric tension of 2.6 pF was established as an appropriate water regime for sweet maize growth.
Key words: Zea mays L. saccharata Sturt., nitrogen, fertilizers, soil water potential, growth conditions
INTRODUCTION
In the tropics, from where sweet maize (Zea mays L. saccharata Sturt.) originates, the most important limiting factors for its growth are water and nitrogen (N) supply (Moser et al. 2006). The crop is grown at latitudes between 50°N and 40°S, and at altitudes 0-3,000 m above sea level (Ghorpade et al. 1998), and to establish sweet maize production in a temperate climate requires research into cultivation practices. Moreover, climate change (i.e. increased temperatures) suggests the possibility of its wider production, which will require further research in areas currently considered atypical for maize (Bavec and Bavec 2002).
Limiting factors for sweet maize production are high temperatures and water stress. High air temperatures (> 38°C) and concurrent water stress decrease yields (Ramadoss et al. 2004). In semi-arid regions, where temperatures are high
and problems with water and irrigation are common, Sari et al. (2000) reported that April is the preferred sowing time for cob and yield quality, and that later sowing in May reduces yield. Oktem et al. (2003) recommended a drip irrigation system with two-day irrigation frequency (100 % of evapotranspiration) to achieve optimum growth of sweet maize in semi-arid regions. Stone et al. (2001) analyzed the drought responses of sweet maize water use, biomass, yield, and yield components and found that yield was strongly related to biomass accumulation (especially to biomass accumulated after brooming). Biomass reduction occurred mainly due to the effects of drought stress on radiation use efficiency, particularly at early growth stages.
When considering the growth of sweet maize, special attention needs to be devoted to N nutrition and possible environmental pollution by N residues after harvesting the crop (Silgram and Shepherd 1999). N residues include the residual soil mineral N (Nmin) and N in crop residues. Using
Correspondence to: E-mail: franci.bavec@um.si
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Sweet maize: response to fertilizers, N rates and soil water
the recommended N rates for vegetables, however, may leave large amounts of residual soil Nmin, especially if crops such as sweet maize are harvested before maturity (Neeteson et al. 1999). Olaniyan et al. (2004) suggested that the use of organo-mineral fertilizers reduces nitrate losses due to leaching and improves soil structure; and that the highest yield and total dry matter were attained at the highest N rate of 120 kg N ha-1. Juntharapthep et al. (2007) found that fermented chicken manure was an efficient fertilizer and produced the highest yields of unhusked, husked and standard unhusked cob for sweet maize (14.25, 10.56 and 13.62 t ha-1, respectively).
Many aspects of plant growth, such as plant photosynthetic activity, N concentration and protein content are affected by drought stress, which also influences nitrate reductase activity in species such as maize (Foyer et al. 1998) and winter wheat (Xu and Yu 2006). Prolonged periods of dehydration stress inhibit photosynthesis of most active mesophyll cells, suppressing metabolism and lowering water use efficiency. Effects on stomatal conductivity are as important as those on photosynthesis (Taiz and Zeiger 2002). Research on maize (Jacob and Lawlor 1992), wheat (Xu et al. 2009) and sweet maize (Xu et al. 2004; Fletcher et al. 2008) has shown that photosynthetic functions depend on many growth factors. Xu et al. (2004) noted that an increase in photosynthetic activity of sweet maize and the quantity of dry matter might be associated with stomatal opening and biochemical activities.
The lack of information on sweet maize cultivation practices limits its introduction into temperate climate zones. For this reason, data obtained from pot experiments provides valuable knowledge regarding the (i) influences of different N rates, (ii) the applied form of fertilizers (i.e. organic or mineral), and (iii) of various water regimes on morphological and yield characteristics, and photosynthetic parameters of sweet maize.
MATERIAL AND METHODS
The effects of different N target values, fertilizer types and water regimes were studied on sweet maize morphological, photosynthesis and yield parameters in a greenhouse pot experiment (University Research Centre Maribor, Slovenia: 46°39'N, 15°41'E and 282 m.a.s.l.) under natural light and temperature conditions. N rates of 0 (control), 0.6 and 2.0 g N pot-1 were calculated similarly to Rodrigues (personal communication), as equivalent approximations of N target values: 70 and 170 kg N ha-1, and control (presowing Nmin). The examined N rates (41 and 141 kg N ha-1) were calculated according to the following formula: N rate (kg N ha-1) = N target value - kg Nmin in the soil (i.e. 29 kg Nmin ha-1). Different organic by-products (pumpkin cake from oil processing with 9 % N, and digestate from biogas production of pig manure with 1.6 % N) and mineral fertilizers (CAN - 27% ammonium nitrate NH„NO„ and ENTEC'26 - 26%
4 3
ammonium nitrate and ammonium sulphate (NH4)2SO4 with nitrification inhibitor (DMPP)) were applied to pots maintained at three water regimes.
A three-factor randomized block design was used. The basic block consisted of four pots (0.14 m in diameter and
0.90 m in height) and was carried out in three replications using sweet maize hybrid 'Gold cup F1' with normal sugar content 'su.
Top soil of sandy loam texture was collected near the greenhouse, homogenized and used to fill the pots. The chemical and physical properties of used soil were: organic matter 11.2 mg g-1, P2O5 52.2 mg kg-1, and available K2O 233.2 mg kg-1. After filling the experimental pots soil (15.8 kg per pot), with a bench mat at the bottom, were placed to the constructed skeleton of building net. The iron net supported the pots and maintained a constant distance of 30 cm between the plants.
Before sowing, Nmin was analyzed (Nmin = nitrate-N + ammonium-N = 8 mg kg-1) in the soil (Scharph and Wehrmann 1975; ISO/DIS 14255 1998), and on this basis the N rate was calculated. Fertilizers were applied before sowing, mixed in the upper 0.1 m of soil in the pot. Soil Nmin was also analyzed at BBCH 75-79 growth stage (milk stage) (Zadoks et al. 1974) and samples were taken from all pots. In total, 300 pots were used. Due to technical limitations (i.e. greenhouse area) the control treatment without added N was analyzed only at the optimal water regime (2.6 pF).
Three days after filling pots, sweet maize was sown into pots (after emergence, thinning reduced the numbers to one plant per pot) and tensiometer tubes (30 cm long) were placed randomly into pots with one tensiometer per treatment (i.e. only in the first replication). After sowing, pots were subjected to the three different soil water regimes. Water regimes were controlled as by Van der Vecken et al. (2003) with tensiometers at soil matric potentials of 2.40 pF (considered as overwatered), 2.60 pF (optimal watered) and 2.80 pF (drought stress) as measured by SMS model 2500 (SDEC, France), where pF units are logarithms of hPa. At BBCH 13-14 stage, all treatments were sustained at optimal water regime (2.6 pF). The tensiometers were placed in pots for daily measurements of the pressure generated in the water column and to calculate the amount of water needed for a given water regime. Tensiometer measurements were performed daily before irrigation. The relationship between matric potential and gravimetric soil moisture content (from wilting point to field capacity) was read from the calculated desorption curve (ISO 11274 1998).
Morphological and yield parameters for all plants were determined at harvest. Measurements included (masses are all in fresh weights): cob mass, plant mass (whole plant), plant green mass (aboveground biomass), root mass, leaf mass, stem mass, broom mass, root length, cob length, cob diameter and plant height.
During the growing season, photosynthetic parameters (photosynthetic rate - A, and stomatal conductance - gs) were monitored by gas-exchange equipment (LCpro+, ACD BioScientific Ltd, UK). Environmental conditions in the leaf chamber were set by LCpro+ equipment. Measurements were made during 10:00-14:00 h on the upper fully expanded leaf at stages BBCH 15-17 (A1 and gs1), 65-69 (A2 and gs2) and 75-79 (A3 and g 3) using the method of Hirasava and Hsiao (1999). They were repeated five times at min-1 intervals and the mean of the five readings was taken as the measured value.
An analysis of variance (ANOVA; P < 0.001, P < 0.01 and P
34
Sweet maize: response to fertilizers, N rates and soil water
< 0.05) for a factorial experiment (fertilizer x N rate x water regime) was performed using the Statgraphic® Centurion (2005) statistical package. Significant differences among treatments were determined using least significant difference (LSD) test at P < 0.05. Pearson's correlation coefficients between morphological and yield parameters, photosynthetic parameters and Nmin were calculated using SPSS 15.0 for Windows statistical package (2005). A quadratic regression was calculated only for some significant properties.
RESULTS AND DISCUSSION Morphological characteristics
In the case of N rates, there were no significant differences in root, leaf and broom mass (Table 1). In contrast, plant mass, green mass, stem mass and plant height significantly increased as N rates were increased. There was a significant effect of N on root length, with significantly longer roots at the lower N rate of 0.6 g pot-1. This indicated the possibility that plants experiencing less N in the soil develop deeper root systems. Similar results were also reported for common maize by Rhoads and Bennett (1990), who found out that crop roots took up nutrients and water from upper levels of the soil under low-water stress or non-stress conditions. Furthermore, based on the measured morphological parameters, pumpkin cake fertilizer appeared to be an acceptable substitute for the N mineral fertilizers. When ENTEC'26 fertilizer, which has
a longer fertilization effect, was compared with either CAN 27 or pumpkin cake, there were no significant differences. Similarly, Guertal (2000) reported results with bell pepper where slow-release fertilizers had no consistent improvement over a soluble N source. The use of digestate in the present study resulted in significantly lower plant mass and plant height, and also in less N residues at harvest compared to the other three fertilizers. These results can be explained by the use of fresh digestate, which can lead to phytotoxic effects or to N loss by ammonia volatilization (Abdullahi et al. 2008; Fuchs et al. 2008).
Water regimes also had a significant effect on all investigated morphological parameters of sweet maize. Except for root length, there was no significant difference between overwatered and optimal water regimes in any morphological parameter. However, all parameters were significantly lower under drought stress conditions. Only root length significantly differed in all water regimes; in comparison to the optimal water regime, there were significantly longer roots in the overwatered condition, and significantly shorter roots for drought stress. In contrast, Kirtok (1998) found in common maize that frequently watered plants produced a shallow root system, whereas occasionally watered plants produced a deep root system. However, in the present study the root mass of overwatered compared to optimally watered plants was not different (Table 1).
Table 1: Effects of water supply and N fertilization on morphological parameters (cob mass-CM; plant mass-PM; plant green mass-GM; root mass-RM; leaf mass-LM; stem mass-SM; broom mass-BM; root length-RL; cob length-CL; cob diameter-CD; plant height-PH) of sweet maize (mean values per plant)
Treatments	CM (g) PM (g) GM (g) RM (g) LM (g) SM (g) BM (g) RL (cm) CL (cm) CD (cm) PH (cm)
Fertilizer (F)	***	**	NS	**	NS	*	***	NS	***	***	***
N rate (N)	*	**	**	NS	NS	**	NS	***	***	***	*
Water regime (W)	**	***	***	***	***	***	***	***	***	***	***
Interaction											
FxN	NS	**	***	***	**	**	*	NS	***	***	***
FxW	NS	NS	***	***	NS	***	***	***	***	***	**
NxW	*	**	**	NS	*	*	NS	NS	**	**	NS
FxNxW	NS	NS	NS	**	NS	*	*	***	*	NS	NS
Fertilizer											
Oil pumpkin cake	85.8 a	210.7 a	124.9	72.3 a	44.2	77.6 a	3.0 a	84.4	5.7 a	1.7 b	155.8 a
Digestate	21.5 b	138.9 c	117.4	58.2 b	39.3	75.3 a	2.8 ab	76.7	1.5 b	0.7 c	131.3 b
CAN 27	61.9 a	181.5 ab	119.7	51.1 b	42.9	74.1 a	2.6 b	84.2	6.6 a	2.1 ab	156.8 a
ENTEC'26	66.2 a	175.4 b	109.2	52.8 b	43.0	64.3 b	1.8 c	85.0	7.0 a	2.3 a	157.9 a
N rate (g pot-1)											
0.60	47.6 b	158.6 b	110.9 b	54.6	40.2	68.1 b	2.6	88.0 a	4.1 b	1.3 b	147.0 b
2.00	70.1 a	194.6 a	124.6 a	62.6	44.5	77.5 a	2.5	77.2 b	6.3 a	2.0 a	153.9 a
Water regime											
Drought stress	30.4 b	115.3 b	84.9 b	20.2 b	27.9 b	55.1 b	1.9 b	70.6 c	1.2 b	0.4 b	114.0 b
Optimal water	74.5 a	210.0 a	135.5 a	79.8 a	49.8 a	82.7 a	2.9 a	82.6 b	7.6 a	2.4 a	170.7 a
Overwatered	71.6 a	204.6 a	132.9 a	75.8 a	49.5 a	80.6 a	2.9 a	94.6 a	6.8 a	2.3 a	166.7 a
*, **, ***, NS: significant at P < 0.05, P < 0.01, P < 0.001, and not significant, respectively
a-c values within column followed by the same letter are not significantly different at P < 0.05 (LSD test).
35
Sweet maize: response to fertilizers, N rates and soil water
Parameters of yield formation
There were significant differences in cob mass for the different fertilizers, N rates and water regimes (Table 1), but the only significant interaction was N rate x water regime (Figure 1). The pumpkin-cake organic fertilizer produced cobs of similar mass than those fertilized by CAN 27 and ENTEC26. Rodrigues et al. (2010) found similar results for tall cabbage where ENTEC26 and urea were not statistically different in dry matter yield and nitrogen use efficiency, in field and pot experiments. In the present study, the digestate resulted in significantly lower values of cob mass, length and diameter compared to the other fertilizers. A similar pattern to cob mass was also found for cob length. Treatment with ENTEC26 resulted in significantly greater cob diameter compared to pumpkin cake and digestate.
The cob mass, length and diameter were significantly affected by different N rates and water regimes (Table 1). There was higher cob mass, length and diameter at the higher N rate (2 g pot-1). Drought stress resulted in significantly lower cob mass, length and diameter in comparison to optimal and overwatered regimes; furthermore, these parameters were similar for the overwatered and optimal water conditions. Zhang and Davies (1989) reported a similar reduction in yield when crops were exposed to water stress for prolonged periods.
There was a strong positive correlation r = 0.778 (P < 0.001) between cob mass and plant mass (Table 2). The significant quadratic regression (y = - 0.014x2 + 0.0954x - 9.95461; R2 = 0.88, P > 0.001) can explain the high influence of plant mass (x) on cob mass (y). These results were similar to those of Stone et al. (2001) who reported that yield of sweet maize is strongly related to biomass accumulation, especially to biomass accumulation after silking. There were moderate correlations between cob mass and root mass (r = 0.529, P < 0.001), and strong correlations between cob mass and plant height (r = 0.775, P < 0.001). Plant mass was also correlated with root mass (r = 0.509, P < 0.001) and with plant height (r = 0.634, P < 0.001).
N rate
Different letters indicate significant differences between treatments (P < 0.05).
Fig. 1: Interactions between examined treatments, water supply (drought stress-W1, optimal water-W2 and overwatered-W3) and N rates (N1-0.6 g pot-1; N2-2 g pot-1) treatments for the cob mass (CM) at sweet maize growth stage BBCH 75-79; parallel is shown control plot at optimal water regime
(CON)
Table 2: Correlations (Pearson) between morphological characteristics (n=72) of sweet maize (plant mass-PM; plant green mass-GM; cob mass-CM; root mass-RM; plant height-PH; root length-RL)
Analyzed parameters
GM
CM
RM
PH
RL
PM	0.777*** 0.778*** 0.509*** 0.634*** 0.213
GM	0.617*** 0.642*** 0.615*** 0.233*
CM	0.529*** 0.775*** 0.196
RM	0.562***	0.421***
PH	0.389*** * significant at 0.05, *** significant at 0.001.
Photosynthetic parameters
Photosynthetic rate (Table 3) was significantly affected by N rate (at BBCH 15-17), by water regime (at BBCH 65-67), and by fertilizer and water regime at harvest (BBCH 75-79). At BBCH 15-17, the measured photosynthetic rate was significantly higher at lower N rate (0.6 g pot-1), but not at brooming and harvest. Zhao et al. (2005) reported that decreased photosynthetic rate of sorghum plants was associated with N deficiency. In comparison to the optimal water regime, there was a lower photosynthetic rate (BBCH 65-69 and 75-79) under drought stress conditions. Photosynthetic rate at BBCH 75-79 was lowest for digestate (4.0 pmol m-2s-1) and was higher for pumpkin cake (5.2 pmol m-2s-1), CAN 27 (6.5 pmol m-2s-1) and ENTEC'26 (8.5 pmol m-2s-1). Investigations of Efthimiadou et al. (2009) under field conditions, showed that photosynthetic rate (66 d after sowing) was significantly higher with application of cow manure at 240 kg N ha-1 compared to barley mulch and poultry manure (both at 35, 70 and 140 kg N ha-1) and mineral fertilizer (240 kg N ha-1). In the present study, of all investigated factors only water regime influenced the stomatal conductance at BBCH 65-67: it was significantly lower in drought stress compared to optimal conditions and also lower in the overwatered regime. Stomatal conductance was not significantly different between N rates and between fertilizer treatments. Similar results were achieved also by Efthimiadou et al. (2009) who investigated organic manures and conventional fertilizer in field conditions.
There were moderate correlations (data not shown) between stomatal conductance and photosynthetic rate at BBCH 1517 (r = 0.553, P < 0.001) and BBCH 65-67 (r = 0.699, P < 0.001), but a weak correlation at BBCH 75-79 (r = 0.332, P < 0.001). Xu et al. (2004) also concluded that leaf stomatal opening for sweet maize was positively related to increases in photosynthetic functions and quantity of plant dry matter. The correlations between photosynthetic parameters (A1-A3 and gs1-gs3) and morphological characteristics were calculated (Table 4).
Correlations between photosynthetic rates and plant height (Table 4) were not strong, but increased during successive growth stages (r = 0.166, r = 0.406, P < 0.001, and r = 0.495, P < 0.001, respectively). Similarly, correlation strength between photosynthetic rate and plant mass, cob mass, cob diameter and cob length with growth increased.
36
Sweet maize: response to fertilizers, N rates and soil water
Table 3: Effects of water supply and N fertilization on Nmin residuals in the soil at harvest, photosynthetic rate (A1-A3 at BBCH 15-17, 65-67 and 75-79, respectively) and stomatal conductance (gs1-3gs1 at BBCH 15-17, 65-67 and 75-79, respectively) of sweet maize (mean values per plant)
Treatments	Nmin	A1	A2	A3	gs1	gs2	gs3
	mg kg-1		|imol m-2s-1			mol m-2s-1	
Fertilizer (F)	***	NS	NS	***	NS	NS	NS
N rate (N)	***	**	NS	NS	NS	NS	NS
Water regime (W)	NS	NS	*	***	NS	*	NS
Interaction							
FxN	***	NS	*	**	NS	NS	NS
FxW	***	NS	NS	***	NS	NS	NS
NxW	***	NS	NS	NS	NS	*	NS
FxNxW	***	NS	NS	*	NS	NS	NS
Fertilizer							
Oil pumpkin cake	56 a	18.0	13.0	5.2 bc	0.12	0.10	0.07
Digestate	6 c	18.8	12.7	4.0 c	0.14	0.10	0.06
CAN 27	49 ab	19.2	15.6	6.5 b	0.13	0.10	0.08
ENTEC'26	43 b	19.4	11.4	8.5 a	0.14	0.09	0.11
N rate (g pot-1)							
0.60	17 b	20.2 a	13.8	5.8	0.14	0.10	0.07
2.00	59 a	17.5 b	12.5	6.2	0.13	0.09	0.08
Water regime							
Drought stress	38	17.6	10.1 b	3.8 b	0.12	0.08 b	0.07
Optimal water	37	19.1	15.7 a	7.6 a	0.13	0.12 a	0.09
Overwatered	41	19.8	13.7 ab	6.6 a	0.15	0.10 ab	0.08
*, **, ***, NS: significant at P < 0.05, P < 0.01, P < 0.001, and not significant, respectively
a-c values within column followed by the same letter are not significantly different at P < 0.05 (LSD test).
Table 4: Correlations (Pearson) between photosynthetic parameters (A1-A3 and gs1-3 at BBCH 15-17, 65-67 and 75-79, respectively) and morphological characteristics, n=72
Analyzed parameters
PH
PM
GM
CM
CD
CL
gsl	0.270*	-0.006	0.083	0.038	0.072	0.064
A1	0.166	0.087	0.258*	0.090	0.133	0.105
gs2	0.356**	0.163	0.209	0.271*	0.263*	0.266*
A2	0.406***	0.256*	0.304**	0.404***	0.362**	0.429**
gs3	0.171	0.124	0.060	0.198	0.238*	0.238*
A3	0.495***	0.387** 0.202		0.589***	0.630***	0.649***
* , **, *** significant at the 0.05, 0.01, 0.001, respectively.
Only correlations between green mass (plant mass without cob) and photosynthetic rate at harvest were lower than at brooming stage. Correlations between stomatal conductance and morphological characteristics were weak; but the values increased to brooming growth stage and then declined by harvest.
Nmin at harvest
Residual Nmin in soil varied significantly with fertilizers and N rate; as well as with interactions of fertilizer x N rate, fertilizer x water regime, N rate x water regime, and fertilizer
x N rate x water regime (Table 3). Residual Nmin values were significantly higher at higher N rates.
The digestate resulted in the lowest Nmin residuals (6 mg N kg-1) at harvest (BBCH 75-79), and can be explained by N immobilization risk (Fuchs et al. 2008). Schievano et al. (2009) explained that immobilization of N in the soil may occur with the use of not-fully matured digestates.
There was significantly higher residual soil Nmin following fertilization using pumpkin cake, CAN 27 and ENTEC'26 (56, 49 and 43 mg N kg-1, respectively); the corresponding converted values were approximately (using 1200 kg m-3 specific mass of the soil) 205, 179 and 154 kg N ha-1. High residual values imply potential for groundwater pollution. Additionally, it is important to account for higher temperatures in the greenhouse compared to field conditions, which can increase mineralization processes in the soil. A laboratory leaching study (Huett and Gogel 2000) showed that controlled-release fertilizers at higher temperatures (30-40°C) had an N-release increased and period was shorter. But, this results lead to more clearly picture in case of interaction fertilizer x N rate (Figure 2). The mineral fertilizers at a higher N rate (2 g pot-1) resulted in higher Nmin residuals compared to the lower N rate (0.6 g pot-1). Especially high Nmin residuals were measured after CAN 27 application in comparison to pumpkin cake and digestate. In general, in the case of mineral fertilizers the lower N rate (0.6 g pot-1) resulted in environmentally acceptable Nmin residuals (in accordance with legislation); and also the use of digestate at both N rates resulted in low Nmin residuals (5 and 6 mg N
37
Sweet maize: response to fertilizers, N rates and soil water
kg-1). Based on a pot and field study with tall cabbage and controlled-release fertilizers, Rodrigues et al. (2010) found that ENTEC'26 was not a fertilizer that prevented N from leaching during winter conditions. Furthermore, in our case no significant differences were observed when water regimes were analyzed.
CONCLUSIONS
The by-product of pumpkin cake as an organic fertilizer had comparable effects on growth (i.e. morphological parameters) and yield parameters (cob mass, length and diameter) of sweet maize as the mineral fertilizers. ENTEC26 as a slow-release N fertilizer showed no advantage for sweet maize in any observed parameter (morphological, yield and photosynthetic parameters) in comparison to pumpkin cake or CAN 27. Fertilizing with a lower N rate (0.6 g pot-1) resulted in significantly lower values of plant mass, cob mass, green mass, stem mass, cob length, cob diameter and plant height, compared to the higher N rate (2 g pot-1); however, there were high soil Nmin residuals at the highest N rate (59 mg N kg-1). There were similarly high Nmin residuals for all investigated fertilizers (56, 49 and 43 mg N kg-1), except for pig manure digestate (6 mg N kg-1). In addition, it is important to account for the higher temperatures in the greenhouse than in field conditions, since they can increase mineralization processes in soil. Furthermore, drought stress was associated with significantly lower values for all measured morphological characteristics in comparison to the optimal and overwatered regimes; however, soil Nmin residuals were not significantly different. The optimal watered regime of 2.6 pF matric potential was confirmed as an appropriate water regime, since the same or significantly higher morphological, photosynthetic or yield parameters were achieved compared to overwatered (2.4 pF) and drought stress (2.8 pF) conditions. Photosynthetic rate was significantly lower at brooming and at harvest under drought stress conditions, in comparison to the optimal and overwatered regimes. Moreover, correlations between photosynthetic rate and yield parameters were weak to moderate, but increased during successive growth stages. This study contributes results and offers opportunities for wider investigations in the field, especially for temperate climates, considering the lack of available sweet maize research results.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the financial support of national projects CRP V4-0472 (Sweet maize - development of new, economic and environmentally acceptable products) and V4-0104 (Organic Vegetable Production) conducted by the Government of the Republic of Slovenia, Ministry of Agriculture, Forestry and Food.
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39
Sweet maize: response to fertilizers, N rates and soil water
Rast in pridelek sladke koruze v odvisnosti od organskih in mineralnih dušikovih gnojil, odmerkov dušika in oskrbe z vodo
IZVLEČEK
Sladka koruza je v evropskih območjih z zmernim podnebjem premalo pridelovana zelenjadnica, njena poraba pa narašča. Za boljše razumevanje načinov oskrbe sladke koruze, je namen lončnega poskusa ovrednotiti vpliv različnih vodnih režimov in odmerkov dušika (N) izračunanih iz ciljnih vrednosti. N v odmerkih 0 (kontrola), 0,6 in 2 g N lonec-1 je bil dodan v obliki organskih ostankov - bučne pogače in digestat iz prašičje gnojevke, ter mineralnih gnojil - CAN 27 in ENTEC26. Obravnavanja oskrbe z vodo so temeljila na izmerjenih talnih matričnih potencialih: 2,8 pF (sušni stres), 2,6 pF (optimalna preskrbljenost z vodo) in 2,4 pF (prekomerna oskrbljenost tal z vodo). V primerjavi z mineralnimi gnojili, so se bučne pogače izkazale za enako učinkovite glede na izmerjeno višino rastlin (155,8 cm), maso storža (85,8 g), zeleno maso (124,9 g) in maso listov na rastlino (44,2 g), oziroma celo kot boljše gnojilo glede na izmerjeno maso korenin (72,3 g) in metlic na rastlino (3,0 g). Parametri pridelka, kot masa (70,1 g), dolžina (6,3 cm) in premer storža (2,0 cm), kakor tudi ostanki mineralnega dušika (59 mg N kg-1) so bili značilno višji pri gnojenju z najvišjim odmerkom N. Statistično nižje vrednosti vrednotenih morfoloških in parametrov fotosintetske aktivnosti (merjeno ob metličenju in spravilu) so bile povezane s sušnim stresom. Matrični potencial 2,6 pF se je izkazal kot najustreznejši za rast sladke koruze.
40
Agricultura 12: No 1-2: 41-47 (2015)
DE
G
DE GRUYTER
OPEN
Variations in leaf total protein, phenolic and thiol contents amongst old varieties of mulberry from the Gorizia region
Tina UGULIN1*, Tamas BAKONYI2, Rebeka BERČIČ2, Andreja URBANEK KRAJNC1
1Faculty of Agriculture and Life Sciences, University of Maribor, Pivola 10, 2311 Hoče, Slovenia, e-mail: andreja.urbanek@um.si 2Faculty of Veterinary Science, Szent Istvân University, Hungâria krt. 23-25, H-1143 Budapest, Hungary
ABSTRACT
Preserving the plant genetic resources of genus Morus is insufficient but undoubtedly vital for conservation of the world's germplasm for our successors. This research was focused on old mulberry varieties from the Gorizia region in Slovenia which were assessed for their contents on crucial metabolites (proteins, phenolics and thiols) in leaves regarding their antioxidant and nutraceutical potentials. Total proteins were measured spectrophotometrically by following the procedure of Bradford, the total phenolic contents were determined using the Folin-Ciocalteu method and thiols were established with monobromobimane fluorescent dye. The presented metabolite screening showed that some of the evaluated genotypes had higher concentrations of glutathione and were superior in contents of proteins and phenolics when compared to the results of other authors and could be propagated as highly recommendable feed for silkworms, and other animals.
Key words: feeds, glutathione, Morus, phenols, proteins
INTRODUCTION
Preserving the plant genetic resources of Morus genus is still insufficient but undoubtedly vital for the conservation of the world's germplast for our successors. Plant material can be preserved in herbariums and museums, while the main strategies of conservation can be divided into in situ conservation, ex situ conservation, in vitro conservation, and DNA banking (Vijayan et al. 2011a). Old varieties are a chance for sustainable leaves production for the growing silk industry. The main method of mulberry propagation is through stem cuttings or grafting of dormant buds (Vijayan et al. 2011b).
Mulberry trees originate from Asia and are currently cultivated in subtropical, tropical, and moderate environments. Morus L. (Moraceae) is a genus that is comprised of 10 to 15 species (Nepal, 2008). After expansion from Venice, sericulture and mulberry cultivation within the Gorizia region of Slovenia started to flourish in the middle of the 16th century (Ipavec 2008). Today sericulture in Slovenia has perished, and with plantations gone, only individual trees can be found, mostly near houses. The number of these types of trees in Slovenia is at the present time unknown. Still,
sericulture is today existent in 70 other countries amongst which the leaders are China, India, Vietnam, Uzbekistan, Brazil, and Thailand.
Old genotypes are interesting for research because of their adaptations to environmental conditions. As a result of naturalisation the classification of cultivars based on morpho-phenological attributes is problematic. Leaf shapes are often diverse, even on the same tree, and are influenced by several factors suchas plant maturity, growth, leaf position on the branch, and period of growth. Mulberry trees can be monoecious or dioecious which is a challenge for phylogeny.
Throughout history when in the cases of drought or when typical fodder wasn't available, mulberry leaves were given to livestock. Fresh leaves were provided to goats, cattle, horses and pigs, although in small quantities. Mulberry leaves are a well-known fodder in sericulture, where it is the sole source because of its suitable nutritional composition which includes proteins, carbohydrates, chlorophyll and carotenoids. These nutritional parameters of mulberry leaves and silk production are directly proportional to each other (Kumar et al. 2010). Leaves have a high protein content (18 to 25 % DM) and high digestibility (75 to 85 %) (Ba et al.2005).
*Correspondence to: E-mail: tina.ugulin@um.si
41
Variations in leaf total protein, phenolic and thiol contents amongst old varieties of mulberry from the Gorizia region
The type of leaves preferred by silkworms are characterized by lower levels of minerals and crude fibers (Krishnaswami et al. 1970). They can also be used in human diet as non-toxic effects on internal organs were found (Srivastava et al. 2003). Regarding the high contents of proteins in mulberry leaves, they have potential as a forage supplement for animals such as sheep (Liu et al. 2001), goats (Bakshi and Wadhava 2007), and rabbits (Martinez et al. 2005).
Different parts of mulberry preparations have been used in traditional phytomedicine mainly due to high concentrationsof antioxidants such as phenolics (Nakamura et al. 2009). Among them phenolic acids and flavonoids possess the highest antioxidant potential. Data also shows that mulberry leaves are abundant in caffeoylquinic acids (6.8 - 8.5 mg/g DM) and flavonols (3.7 - 9.8 mg/g DM) as recorded by Sánchez-Salcedo et al. (2015). Because of the high antioxidant properties of Morus species, these leaves have the potential of being used as dietary supplements and food products for human consumption also. On the grounds of the high phenolic content of mulberry extracts there is a potential exploitation of a phytochemical nature.
Morus alba is widely valued in traditional medicine, where it is known by the name Mori folium, for diabetes, protecting the liver, and lower blood pressure (Kim et al. 2014). Confections containing Morus alba extracts contribute to the prevention and qualities of life for pre-diabetic and diabetic patients (Nakamura et al. 2009; Ma et al. 2014). Mulberry leaf polyphenols possess antiatherogenesis effects that could be studied further (Yang et al. 2011). Yang et al. (2011) came to the conclusion that a single administration of a water extract from leaves of M. alba lowers systolic blood pressure in a dose-dependent way. Another study showed that a long-term administration of M. albaleaves resulted in decrease in body weight and hepatic lipid accumulation (Oh et al. 2009).
Part of the main objective of the study was to screen the biochemical contents of important primary (proteins) and secondary metabolites (phenolics, cysteine, glutathione) in leaves of local mulberry varieties obtained from the Gorizia region, in order to test their relevance as animal feed.
MATERIALS AND METHODS
Plant material
The samples of mulberry leaves were collected from 22 selected old mulberry genotypes from the Gorizia region in Slovenia. Five fully developed sun-exposed leaves (7th leaf from apex) were gathered randomly from each tree on August 20th 2015 and used as one sample. The mulberry trees were of old genotype origin with perimeters ranging from 80 to 370 cm. The meteorological information is given in the Table 1.
Meteorological information was obtained from the nearest weather station BILJE on August 20th 2015 from 07:00 to 21:00 hours: Temperature: 16.8 - 26.9 °C; Relative humidity: 48 - 99 %; Wind direction: NE, W, WNW; Wind speed: 0.6 - 2.1 m/s.
Table 1: Coordinates of mulberry habitat		
Sample number	Coordinates	Additional information
1 - 6	45.89846038818359, 13.61314487457275	Miren 91
7	45.88313293457Q31, 13.61183166503906	Vrtoče
8 - 9	45.88530731201172, 13.60030746459961	Miren 219
W	45.88692092895508, 13.6Q146427154541	Miren 192
11	45.9Q574645996Q94, 13.61686134338378	Njiva pri Vrtojbi
12	45.90725708007812, 13.61715316772461	Njiva pri Vrtojbi B
13	45.9Q815353393555, 13.62518024444580	Njiva pri Vrtojbi C
14	45.91115126082022, 13.6261641791469Q	Njiva pri Vrtojbi D
15	45.9132Q419311523, 13.62628936767578	Njiva - Krožna cesta
16	45.91765213Q12695, 13.63195Q37841796	Vrtojba
17	45.89608383178711, 13.6445894241333Q	Bilje 149
18 - 2Q	45.89556884765625, 13.6278219223Q224	Pot Bilje -Miren
21 - 22	45.892219543457Q3, 13.63Q835533142Q9	Bilje - pri vodnjaku
Sample preparation for biochemical analysis
Immediately aftercollection, samples were stored in liquid nitrogen. Afterwards they were transferred into the freezer at -80 °C. The tissue samples were subsequently lyophilized and ground. The prepared samples were then stored in air tight vials at -20 °C prior to biochemical analysis.
Extraction and determination of total protein content (tPr)
Total proteins were determined by using the Bradford reagent following the procedure of Bradford (Bradford 1976). The standards were prepared with Bovine Serum Albumine (BSA). TRIS/HCl (1 ml, 0,1 M) (Douchefa) was added to 25 mg of each sample. After being vortexed for 1 min, the samples were centrifuged for 10 min at 14000 RPM. 80 ^l of water was added to the 20 ^l of mixture. Bradford reagent (900 ^l) (Sigma-Aldrich) was added to the samples, standards, and the reference blank. The mixture was then vortexed for 1 min and proteins were spectrophotometrically determined at 595 nm.
42
Variations in leaf total protein, phenolic and thiol contents amongst old varieties of mulberry from the Gorizia region
Extraction and determination of total phenolic content (tPh)
The total amount of phenolic compounds was determined using the Folin-Ciocalteu method following the procedure of Ainsworth and Gillespie (2007). Briefly, 1.5 ml of 95 % methanol was added to the 25 mg of each lyophilized sample. After 5 min of homogenisation at 30 Hz in an ultrasonic bath, the samples were centrifuged at 4 °C and 14000 RPM. Subsequently 100 ^l of supernatant was pipetted and 200 ^l of F-C reagent (10 %) (Merck) was added and vortexed. After exactly 3 minutes 800 ^l of 700mM Na2CO3 (Sigma-Aldrich) was applied and the mixture was maintained at room temperature in the dark for 120 min. The absorbance was measured at 765 nm against a reagent blank using the Varian Cary UV/VIS spectrophotometer. Gallic acid (Sigma-Aldrich) was used as the reference standard. The total phenolic content was expressed as mg of gallic acid equivalents per gram of each extract on dry basis (mg GAE/g DM).
Extraction and determination of thiols (CYS, GSH)
For the glutathione and cysteine analysis a method with the thiol-specific fluorescence dye monobromobimane was used as described by Tausz et al. (2003). HCl (2 ml of 0.1 M, 32 %) (Sigma-Aldrich) was added to 60 mg PVP (Sigma-Aldrich) and after being allowed to rest overnight the mixture expanded. PVP was used here as a method for excluding any disruptive phenols. 40 mg of lyophilizedsample was added to the mixture and homogenized with Ultra-Turrax for 20 sec and afterwards centrifuged for 15 min at 3000 RPM at 4 °C. For the extraction of reduced thiol forms (GSH and CYS) 280 [l of each extract was incubated along with 420 [l of CHESS puffer [5,2 g CHES (Sigma-Aldrich) + 100 ml bi-distilled water + 40 mg EDTA (Sigma-Aldrich) with the pH 9.3) and 70 [l of 5mM DDT [19,2 mg DTT (Fluka) + 25 ml bi-distilled water] solution. After one hour of incubation thereaction started with the application of 50 [l of 8mM monobromobimane[25 mgmonobromobimane (Invitrogen) + 12 ml acetonitrile (Sigma-Aldrich)], followed by 15 min incubation in the dark at room temperature. The derivatisation was stopped with the addition of 600 [l of methansulphonic acid (0.25 %, v/v) (Merck). The suspension was centrifuged for 45 min at 14000 RPM at 4 °C and ultimately 1 ml of each extract was pipetted into vials.
The separation and determination of thiols was carried out using the gradient method, namely, liquid chromatography on the HPLC system Waters 2695, Waters 2475 Multi Fluorescence detector (excitation: 380 nm; emission: 480 nm), column Spherisorb S5 ODS2 25 x 4.6 mm, column temperature 23 ± 1 °C. Solvent A: acetic acid (0.25 %, v/v) in water with the addition of methanol (5 %, pH 3.9) (Sigma-Aldrich). Solvent B: methanol (90 %, v/v) in water with the addition of acetic acid (0.2 %, v/v) gradient: 5 % solvent B to 15 % solvent B in 20 minutes, 100 % solvent B for 6 minutes, and 5 % solvent B for another 8 minutes. Flow rate was 1 mL min-1.
Statistical analysis
The results of biochemical analyses represented the means and standard deviations (S.D.) of three replicate measurements of each sample. The results were evaluated by one-way ANOVA using the program IBM SPSS Statistics 22. Additionally, correlation between secondary metabolites was analyzed. The differences between the samples were compared with the Duncan test at p < 0.05. Significant differences were indicated by different lowercase letters (a-k).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Sample number
Fig. 1: Total proteins' concentrations
RESULTS AND DISCUSSION Total protein content
Amino acids represent a primary class of nutrients. The highest levels of total protein content were analyzed in samples 21, 15 and 22, and the lowest in sample 8, as shown in Figure 1. The protein contents were determined within a range from 35 to 126 mg/g DM.
Kandylis et al. (2008) performed a study of the crude protein content in mulberry leaves with concentrations reaching 200 g/kg DM. The major protein fractions are represented by prolamins (44.1 %) and albumins (11.1 %) followed by globulins (9.7 %) and glutelins (8.5 %). It is known that the total protein contents change with mulberry leaf aging (Matei et al. 2006). As the leaves' content of protein decreases with maturity, it is possible this is the source of differences in concentrations between our study and the one conducted by Kandylis. In another study, it was found that amino acids' content increase during leaf maturity thought the differences were insignificant. The most represented amino acids were reported to be glutamine, asparanin and leucine (Yao et al. 2000).
Total phenolic content
Phenolics are a group among three main categories of secondary plant compounds. Mulberries possess antioxidant properties, mainly because of the content of phenolic compounds (Thirumalaisamy 2009, Memon et al. 2010, Radojkovic et al. 2012, Flaczyk et al. 2013).The DPPH radical-scavenging potential was determined to have a positive correlation (R = 0,803) with the amount of total phenolics (Memon et al. 2010). In leaves of M. alba the main phenolic
43
Variations in leaf total protein, phenolic and thiol contents amongst old varieties of mulberry from the Gorizia region
components were identified as chlorogenic acid, followed by caffeic, vanillic, sinapic, ferulic and gallic acid. The flavonols fraction contains rutin, quercetine, and kaempferol. Meanwhile, Radojkovic et al. (2012) determined ferulic acid as the predominant phenolic compound in Morus alba leaf extract, followed by rutin, sinapic, chlorogenic and gallic acid. Quercetine was not detected. However, in another study conducted by Katsube et al (2005) the predominant flavonol glycoside was indeed quercetine. Mulberry leaves are free of tannins, as reported by Singh and Makkar (2002).
In the presented study, the maximum amount of total phenolic content was documented among samples 17, 11, 19, 18 and 7, where the concentration reached 15.7 g GAE/100 g extract. The lowest levels were analyzedin samples 14, 13, 5, 4, 10, 2, 6, 3, 22 and 21, as shown in Figure 2.
Our results are similar to those reported by Flaczyk et al. (2013), who analyzed total concentration of phenolic compounds equal to 14.4 g GAE/100 g in M. alba leaf extracts. In another study a much lower total phenolic concentration was determined in M. alba leaves reaching a maximum value of 4.56 % (Thirumalaisamy 2009). Some of our genotypes expressing higher tPh contents could be valuable sources of genes in future breedings. Additional research is required focused on single phenolic compounds in order to determine the predominant phenols and their concentration within each genotype.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Sample number
Fig. 2: Total phenols content
Reduced glutathione and cysteine content
Glutathione is the primary low-molecular-weight thiol in most cells. Ordinarily, glutathione is mostly present in its reduced form (GSH), which was measured in this study, with only a small part present in its oxidized state (GSSH) (Dixon et al. 1998). In the absence of stress, plant tissues, including leaves, usually maintain a ratio between GSH:GSSG of a minimum of 20:1 (Noctor et al., 2011). GSH is crucial for plant development by influencing critical functions in embryo and meristem development (Frottin et al. 2009).
Statistically significant differences in the reduced glutathione and cysteine content were determined (p < 0.05). The data was then grouped into homogeneous subsets, 10 groups for cysteine and 11 for glutathione, as shown in Figure 3. The highest rate of cysteine was measured in sample 15 reaching almost 73 nmol/g DM and the lowest consisted in the samples 16, 17, 14, 7 and 8, with concentrations of 24nmol/g. The highest reduced glutathione concentrations were found in samples 5 and 3, with levels of 2000nmol/g,
while the lowest levels were found among samples 8, 16, 17, 14, 10 and 19, with concentrations above 700 nmol/g.
In comparison with beech trees (Fagus sylvatica L.) mulberries have manifold higher concentrations of glutathione (Herbinger et al. 2005).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Sample number
Fig. 3: Cysteine and reduced glutathione concentrations
Correlation between studied biochemical components
The correlation was calculated with the Pearson correlation coefficient, as shown in Table 2. Glutathione is made up of the amino acids glutamine, cysteine and glycine so a high correlation factor between GSH and cysteine was expected. Statistically significant correlation was documented between glutathione and cysteine, with the coefficient reaching 0.707. A lower but statistically significant link was noted between glutathione and proteins, with the coefficient of 0.293. The correlation between cysteine and proteins was 0.241, which was not statistically significant.
Phenolics are known as important antioxidants. As a result of scavenging activities phenolics become oxidized to phenoxyl radicals. The regeneration of phenoxyl radicals can be carried out via ascorbate-glutathione cycle (Szôllôsi 2014). However, the expected correlation between phenols, glutathione and cysteine was not statistically important, as the coefficient between the phenols and thiols was - 0.204 and -0.010. respectively. Correlation between proteins and phenols was not expected.
CONCLUSION
The presented metabolite screening in leaves of old local mulberry varieties (M. alba) from Gorizia region showed that some evaluated genotypes expressed high contents of metabolites. Samples 21, 15 and 22 contained the highest protein concentrations, samples 17, 11, 19, 18 and 7 contained the most phenols, sample 15 contained the highest rate of cysteine, and samples 5 and 3 contained the highest reduced glutathione concentrations. The nutritively richer varieties could be propagated as highly recommendable feed
44
Variations in leaf total protein, phenolic and thiol contents amongst old varieties of mulberry from the Gorizia region
Table 2: Correlation between studied biochemical components
		GSH [nmol/g]	Proteins	CYS [nmol/g]	Phenols
	Pearson Correlation	1	,293*	,707**	-,204
GSH [nmol/g]	Sig. (2-tailed)				
			,037	,000	,151
	Pearson Correlation	,293*	1	,241	-,032
Proteins (mg/g)					
	Sig. (2-tailed)	,037		,089	,772
	Pearson Correlation	,707**	,241	1	-,010
CYS [nmol/g]					
	Sig. (2-tailed)	,000	,089		,942
	Pearson Correlation	-,204	-,032	-,010	1
Phenols (mg/g)					
	Sig. (2-tailed)	,151	,772	,942	
. Correlation is significant at the 0.05 level (2-tailed). *. Correlation is significant at the 0.01 level (2-tailed).
for silkworm and other animals. These conclusions may be a stepping stone for selecting cultivars with desired nutrient profiles for breeding.
ACKNOWLEDGMENT
This research was funded by the Slovenian Research Agency (ARRS, N1-0041). The authors thank Mateja Felicijan and Anja Ivanus for laboratory assistance. The authors also thank the reviewers for valuable comments and suggestions.
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Variations in leaf total protein, phenolic and thiol contents amongst old varieties of mulberry from the Gorizia region
Spremenljivost vsebnosti skupnih proteinov, fenolov in tiolov, v listih starih genotipov murv v Goriški regiji
IZVLEČEK
Ohranjanje genskih virov rodu Morus je nezadostno, čeprav je nedvomno bistvenega pomena za ohranjanje svetovne zapuščine za naše naslednike. Ta raziskava temelji na določitvi ključnih metabolitov (proteinov, fenolov in tiolov) v listih starih genotipovmurv iz Goriške regije v Sloveniji z namenom proučitve antioksidativne aktivnosti in hranilne vrednosti. Skupni proteini in fenoli so bili določeni spektrofotometrično s pomočjo Bradfordove in Folin-Ciocalteu metode, tioli so bili določeni s pomočjo HPLC po predhodnem markiranju s fluorescenčnim barvilom monobromobimane. Rezultati določitve metabolitov kažejo, da imajo nekateri genotipi visoko vsebnost glutationa,v primerjavi z rezultati drugih avtorjev so superiorni v vsebnosti proteinov in fenolov in bodov nadaljnjih raziskavah razmnoženi z namenom uporabe listov za krmo sviloprejk in ostalih živali.
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