Impact of Conventional and Integrated Management Systems on the

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Impact of Conventional and Integrated Management Systems on the Water-Soluble Vitamin Content in Potatoes, Field Beans and Cereals Sabine Freitag, Susan R Verrall, Simon DA Pont, Diane McRae, Julia Anne Sungurtas, Raphaelle Palau, Cathy Hawes, Colin J Alexander, William J Allwood, Alexandre Foito, Derek Stewart, and Louise Shepherd J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03509 • Publication Date (Web): 19 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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Journal of Agricultural and Food Chemistry

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Impact of Conventional and Integrated Management Systems on the

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Water-Soluble Vitamin Content in Potatoes, Field Beans and

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Cereals

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Sabine Freitaga,*, Susan R. Verralla, Simon D.A. Ponta, Diane McRaea, Julia A. Sungurtasa,

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Raphaëlle Palaua, Cathy Hawesa, Colin J. Alexanderb, J. William Allwooda, Alexandre Foitoa,

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Derek Stewarta,c, Louise V.T. Shepherda

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a

Environmental and Biochemical Sciences, The James Hutton Institute, Invergowrie, Dundee

DD2 5DA, UK.

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b

Biomathematics and Statistics Scotland, Invergowrie, Dundee, DD2 5DA, UK.

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c

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UK.

School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, EH14 4AS,

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*To whom correspondence should be addressed: Tel: + 44 (0)1382 568919; Email:

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[email protected] 1 ACS Paragon Plus Environment


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Abstract

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The reduction of the environmental footprint of crop production without compromising crop

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yield and their nutritional value is a key goal for improving the sustainability of agriculture. In

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2009, the Balruddery Farm Platform was established at The James Hutton Institute as a long-

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term experimental platform for cross-disciplinary research of crops using two agricultural

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ecosystems.

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integrated management systems, and analyzed for their water-soluble vitamin content.

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Integrated management, when compared with the conventional system, had only minor effects

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on water-soluble vitamin content, where significantly higher differences were seen for the

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conventional management practice on the levels of thiamine in field beans (p < 0.01), Spring

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barley (p < 0.05) and Winter wheat (p < 0.05), and for nicotinic acid in Spring barley (p < 0.05).

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However, for all crops, Variety and Year differences were of greater importance. These results

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indicate that the integrated management system described in this study does not significantly

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affect the water-soluble vitamin content of the crops analyzed here.

Crops representative of UK agriculture were grown under conventional and

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Key Words: barley (Hordeum vulgare L.), field beans (Vicia faba L.), integrated management,

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Liquid Chromatography-Triple Quadrupole-Mass Spectrometry, potato (Solanum tuberosum L.),

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water-soluble vitamins (WSVs), wheat (Triticum aestivum L.). 2 ACS Paragon Plus Environment

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Introduction

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UK1 and EU2 agricultural policies and strategies are geared towards a shift to a more sustainable

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use of resources and conservation of farmland biodiversity.

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understand the impact of the environment, crop management and soil fertility on both the yield

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and nutritional quality of economically important crops in Scotland. Currently, the long-term,

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systems-level impact of changes in management, especially in terms of sustainable treatment

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strategies, has been little studied.

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management whilst maintaining crop yield and nutritional quality. In 2009, The James Hutton

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Institute set up a long-term experimental platform3 for cross-disciplinary research on the use of

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both integrated and conventional management systems in agricultural ecosystems. The platform

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is based on a framework for designing and testing cropping systems that hopes to optimise the

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balance between crop yield and product quality on one hand, with biodiversity and ecosystem

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services on the other (in essence, attempting to assess the balance between economic and

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environmental demands). The aim is to reduce the environmental footprint of crop production

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by minimising the use of fossil fuel derived inputs and maximising the benefits from renewable

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resources. The long-term, whole-systems approach adopted at the platform is essential if the

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potential conflicts between food production and environmental health are to be reconciled. So

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far, much of the literature has focused on the effect of organic versus (vs.) conventional

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cultivation systems on nutritional value4-9, but also yield and cellular processes9-12 mainly in

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potatoes, whilst the impact of lower input (sustainable, organic, integrated) farming practices on

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crop nutritional value has rarely been investigated13,14. Conventional, integrated, sustainable and

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organic cultivation strategies have different goals in relation to crop yield, land and pesticide use,

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and also environmental impact. While conventional agricultural practices utilize high yield crop

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varieties, chemical fertilizers and pesticides, irrigation and mechanization, sustainable farming

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agricultural practices are designed to promote environmental health and the social and economic

Therefore, it is important to

There clearly is a need for balancing environmental

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equity of a region. Sustainable agriculture practices are hard to define as conditions can vary

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greatly depending on the crop, environment, and issues important to a region13.

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The term “nutritional value” as such has been defined as the chemical composition of food, in

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particular the amounts of key compounds that are essential for functioning of human

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organisms15. Vitamins are a broad group of organic bioactive compounds that are minor but

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nutritionally-essential constituents of food. Generally, vitamins can be divided into fat soluble

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(vitamin A, D, E and K1) and water-soluble vitamins (WSVs), which include the B-group

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vitamins thiamine (B1), riboflavin (B2), pyridoxine (B6), nicotinic acid (B3), pantothenic acid

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(B5) and vitamin C. These act as coenzymes, and are therefore essential for a range of different

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metabolic processes16. Most vitamins, apart from vitamin D and nicotinic acid, are essential in

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the human diet as the body cannot synthesize them. With the exception of vitamin B12, only

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plants have the ability to synthesize B vitamins17. Post-harvest processing (such as drying,

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storage, dehulling, milling, soaking, blanching, fermentation and cooking) can impact the levels

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of WSVs in various crops18,19, however, the number of studies on the effects of pre-harvest

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conditions (particularly different agricultural management practices) on content of WSVs have

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been limited. Most studies in the literature, on the impact of different cropping systems on WSV

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contents, have focused on vitamin C content in some fruits, vegetables and eggs8,20-24. Whilst

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vitamin C is generally the most studied vitamin, little data have been published on other B

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vitamin levels in food crops.

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The major challenge previously in quantifying WSVs has been sensitivity, as the low levels in

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crops (such as cereals) require very sensitive methods25, and their simultaneous quantification of

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these diversely different chemistries.

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quantification of single vitamins, e.g. Martins-Junior et al.26, and methods range from

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microbiological assays17, Biosensor/ELISA27 to chromatographic procedures with UV or

Previous methods have mainly focused on the


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fluorescence detection28,29. These extraction and detection methods have several drawbacks

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including the number of B vitamins quantified, lack of precision and their sensitivity. Only

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recently have advances been made regarding increased sensitivity and simultaneous detection of

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WSVs in various food matrices ranging from maize flour, tomato pulp, kiwi to semi-coarse

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wheat flour, wheat bread and toasted wheat bread30,31. Liquid chromatography-triple quadrupole

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mass spectrometry has been optimized as a rapid procedure for multivitamin quantification in

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combination with an optimized extraction procedure30,31. For this study, a method by Nurit et

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al.31 has been adapted to investigate whether our integrated management system impacts upon

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the content of five WSVs in a range of different crop matrices - including field beans (Vicia faba

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L.), Spring and Winter barley (Hordeum vulgare L.), Winter wheat (Triticum aestivum L.) and

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potato (Solanum tuberosum L.) – the latter of which was also analyzed for vitamin C content.

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Material and Methods

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Experimental Design and Preparation of Sample Material

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The Balruddery Farm platform, Dundee3 comprises a 42 hectare (ha) contiguous block of six

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arable fields, north-east Scotland (56.48 latitude-3.13 longitude). Balruddery Farm is a 178 ha

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arable farm, 67 to 163 m above sea level on the south facing slopes of the Sidlaw Hills. The

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farm is typical of temperate Atlantic maritime arable environments, with an average annual

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rainfall of 800 mm, an average annual accumulated temperature of 1100-1375 day oC (above 5.6

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o

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(2.6-4.4 metres second-1 wind speed) and has moderate winters of 50-110 day oC of accumulated

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frost. The soils are imperfectly draining Balrownie Series32 with an average pH of 5.7. Topsoil

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depths range from 25-40 cm, textures from sandy loam to sandy silt loam and stone contents of

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10-20 % volume. Each of the six fields are divided in half, and the integrated and conventional

C) and a mean annual potential water deficit of 50-75 mm. The area is moderately exposed



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management systems randomly allocated to each field half in 2010, at the start of the first

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rotation (Figure 1)3. These systems then remain in place for the duration of the experiment to

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allow detection of a build-up in response to cropping system over time.

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management system is a composite treatment, including tram-line management in cereals and

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tied-ridging in potatoes to reduce soil, water and nutrient loss33; non-inversion tillage to improve

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physical structure and decrease nutrient losses34; green waste compost addition and crop residue

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incorporation to build-up soil carbon and improve physical structure35; green cover (forage

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radish) over Winter before potato to reduce nitrogen (N) losses and increase phosphorous (P)

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uptake36; clover under sowing of Spring barley crops for additional renewable N input to the

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rotation37; lower doses of artificial N fertilizer (taking approximately 75 % of the standard rate as

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a reasonable starting point for this site, based on expert agronomic advice, with further

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reductions planned as soil fertility improves) to reduce environmental footprint, leaching and

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emissions38; lower herbicide dose and alternative chemicals to encourage a diverse weed

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understorey, aiming at approximately 10 % ground cover of non-competitive dicotyledonous

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weeds39, and threshold crop protection applications based on the Home-Grown Cereals Authority

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(HGCA), now the Agriculture and Horticulture Development Board (AHDB) dose response

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curves40 (Table 2). The crop rotation is potato followed by Winter wheat, Winter oilseed rape,

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Winter barley, field (Spring) beans and Spring barley. These crops were selected as typical for

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the Tayside farming region and representative of the most common cropping systems in Scotland

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(and much of the UK). Winter crops are sown in late summer/autumn (August-October) and

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harvested the following summer (July-September). Spring crops are sown between March and

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May, and harvested August-September of the same year. Within each half-field, five different

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varieties of each crop were sown to assess variety-specific responses to the change in

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management systems. For each crop, one variety was selected which was an industry standard,

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providing a comparator to annual UK performance. The remaining varieties were selected for


The integrated

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specific environmental traits such as disease resistance, resource use efficiency and weed

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tolerance3 (Table 1). Table 1. Summary of crops, and their varieties, used for the analysis of water-soluble vitamins over five years.

Crop

Potato

Field Beans

Spring Barley

Winter Barley

Winter Wheat

Year 1

Year 2

Year 3

Year 4

Year 5

2010/2011

2011/2012

2012/2013

2013/2014

2014/2015

Cabaret

Cabaret

Cabaret

Cabaret

Cabaret

Lady Balfour

Lady Balfour

Lady Balfour

Lady Balfour

Lady Balfour

Maris Piper

Maris Piper

Maris Piper

Maris Piper

Maris Piper 1

Mayan Gold

Mayan Gold

Mayan Gold

Mayan Gold

Maris Piper 2

Vales Sovereign

Vales Sovereign

Vales Sovereign

Vales Sovereign

Vales Sovereign

Ben

Ben

Ben

Babylon

Babylon

Fuego

Fuego

Fuego 1

Boxer

Boxer

Maris Bead

Maris Bead

Fuego 2

Fanfare

Fanfare

Pyramid

Pyramid

Pyramid

Fuego

Fuego

Tattoo

Tattoo

Tattoo

Pyramid

Pyramid

4-Component Mix

4-Component Mix

4-Component Mix

4-Component Mix

4-Component Mix

Concerto

Concerto

Concerto

Concerto

Concerto

Optic

Optic

Optic

Optic

Optic

Waggon

Waggon

Waggon

Waggon

Waggon

Westminster

Westminster

Westminster

Westminster

Westminster

4-Component Mix

4-Component Mix

4-Component Mix

4-Component Mix

4-Component Mix

Flaggon

Cassata

Cassata

Cassata

Cassata

Retriever

Retriever

Retriever

Retriever

Retriever

Saffron

Saffron

Saffron

Saffron

Saffron

Sequel

Sequel

Sequel

Sequel

Sequel

Alchemy

Alchemy

Alchemy

Alchemy

Alchemy

Consort

Beluga

Beluga

Beluga

Beluga

Istabraq

Consort

Consort

Consort

Consort

Viscount

Istabraq

Istabraq

Istabraq

Istabraq

Zebedee

Viscount

Viscount

Viscount

Viscount

Where Bold text denotes the industry standard.

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For Spring barley, the same four varieties, and a four-component mix (4-Comp Mix), comprising

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each of those varieties, were grown over 2011-2015. However, for the other crops, adjustments 7 ACS Paragon Plus Environment


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had to be made as to which varieties were grown in each year, depending on seed availability.

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For example, the potato variety Mayan Gold was not available for growth in 2015, however, no

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alternative variety was substituted, rather Maris Piper (the industry standard) was grown in two

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adjacent plot strips (Table 1). For Winter wheat in 2011, Beluga was not available, and Zebedee

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was grown in its place. Similarly, for Winter barley in 2011, Cassata was not available and

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Flaggon was grown in its place. This had implications for the 4-Comp Mix in 2011, as it was not

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comparable with the 4-Comp Mixes generated for Winter barley in 2012-2015. Field beans were

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more complex, where only two varieties – Fuego and Pyramid, were consistently grown over

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2011-2015. Two of the varieties – Ben and Tattoo were only grown over 2011-2013. Maris

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Bead was only grown over 2011-2012. Finally, Babylon, Boxer and Fanfare were available for

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2014-2015. This meant that in total, over the five years, eight varieties of field beans were

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grown. The above has been considered, and factored into the resulting statistical outputs, which

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will be explained in the Statistical Analysis section.

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Cereals and potatoes were harvested from 1 m x 1 m quadrats at five, fixed Global Positioning

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System (GPS) locations in each variety strip as indicated in Figure 1. With regards to the field

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beans, the GPS locations were not physically accessible in the standing crop without causing pod

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shatter and yield loss. Therefore, after each variety strip was harvested, five hand-sampled

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aliquots were collected (providing five technical, rather than spatial, replicates) for WSV

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analysis.

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Following harvest, the sample material was prepared as follows: the three cereal crops were

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dried down to industrial standards (10-15 % moisture still present), threshed with a small

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combine harvester, and then graded (barley: sieve size of 2.5 mm; wheat: sieve size of 2.25 mm).

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The seeds were then milled with Retch ZM 200 ultra-centrifugal mill (Tecator Udy, sieve size


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0.5 mm). The milled powders were then individually packed in polyethylene bags (VWR, UK)

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and stored at -20 °C until required for analysis.

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After the potato harvest, the potato tubers were stored in the dark at ambient temperature for a

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minimum of one week to facilitate skin set - the common UK post-harvest practice. For each

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replicate (GPS point), four to five average-sized potato tubers, with a combined fresh weight of

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approximately 800 g, were selected, washed by hand, and each tuber then transected into eight

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segments. Two diametrically opposed segments (taken to provide a representative sample of the

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whole tuber) were taken from each tuber, and the opposite eighths from the tubers per replicate

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combined, then flash frozen in liquid N and stored at -20 °C. The frozen material was then

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freeze-dried for five days, then the dried material milled using a Retch ZM 200 ultra-centrifugal

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mill (Tecator Udy; 0.5 mm sieve). The milled potato powders were then individually packed in

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polyethylene bags (VWR, UK) and stored at -20 °C (in the dark) until required for analysis.

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Five ‘replicate’ aliquots of field beans, each comprising 60 beans, were flash frozen in liquid N,

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stored at -20 °C, freeze dried overnight, then milled as per the potatoes. The milled field bean

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powders were then individually packed, as for potato, and stored at -20 °C (in the dark) until

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required for analysis.

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Chemicals and Reagents

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All chemicals used for the present study were of analytical grade (purity > 98 %). Analytical

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standards of thiamine hydrochloride, nicotinic acid, pyridoxine hydrochloride, pantothenic acid,

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and riboflavin were obtained from Scientific Laboratory Supplies Ltd (Newhouse, UK).

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Individual stock solutions for all five vitamins were prepared in 50:50 (v/v) acetonitrile/water (4

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mg mL-1). A stock solution of all five vitamins was prepared in 50:50 (v/v) acetonitrile/water 9 ACS Paragon Plus Environment


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with a concentration of 100 µg mL-1. The labelled internal standard pantothenic acid-13C3,15N

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hemicalcium salt was obtained from LGC Standards (Teddington, UK). Thiamine-4-methyl-13C-

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thiazol-5-yl-13C hydrochloride and pyridoxal-methyl-d3 were purchased from Sigma Aldrich

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(Dorset, UK). Internal standard solutions were prepared by dissolving 1 mg in 1 mL 50:50 (v/v)

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acetonitrile/water. Prior to batch extraction, a mix of all three internal standard solutions was

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prepared resulting in a final concentration of 13.6 mg mL-1. Sodium acetate trihydrate, glyoxylic

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acid monohydrate, L-glutathione reduced, ethylenediaminetetraacetic acid (EDTA), sodium

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hydroxide, iron(II) sulphate heptahydrate, formic acid and glacial acetic acid were purchased

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from Fisher Scientific (UK, Analytical Grade). Tris(2-carboxyethyl)phosphine hydrochloride

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(TCEP), metaphosphoric, sulphuric and ascorbic (AsA) acids were purchased from Sigma

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Aldrich (Dorset, UK). HPLC grade acetonitrile was purchased from VWR (West Sussex, UK).

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Ultrapure water (18.2 MΩ.cm) was obtained from an Elga Purelab-Option Q System (High

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Wycombe, UK).

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Quantification of Vitamin C

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The vitamin C content of freeze-dried potato powders were quantified as follows: 100 mg of

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powder was weighted into 2 mL microfuge tubes and resuspended in 1 mL of 5 % (w/v)

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metaphosphoric acid containing 5 mM TCEP, which acts as a reducing agent by converting

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dehydroascorbate (DHA) to AsA. Consequently, results are presented as total vitamin C (total

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AsA). The suspension was vortexed for 10 seconds and transferred onto a blood rotator for 30

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min at 5 °C. Following this, the suspension was centrifuged at 5 °C for 10 min. The supernatant

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was then transferred into a clean 2 mL microfuge tube, and the remaining pellet re-extracted as

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before. Both extracted supernatants were combined, centrifuged to pellet any remaining debris,

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and the supernatant transferred into 0.3 mL transparent polypropylene-short threaded High-

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Performance Liquid Chromatography (HPLC) micro-vials sealed with a 9 mm polypropylene 10 ACS Paragon Plus Environment

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screw cap (VWR, UK), and subjected to HPLC (ASI-100 autosampler, and Ultimate 3000 pump)

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coupled to a UV-Visible detector (UVD340U, Dionex, ThermoFisher SCIENTIFIC, UK).

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Autosampler and column temperature were maintained at 4 °C and 50 °C respectively. 20 µL of

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sample was injected onto an ICSep COREGAL-64H column (ChromTech, USA), with the

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dimensions of 7.8 x 300 mm and particle size of 10 µm and cross linkage of 6.4. An isocratic

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run of 30 min was applied with a mobile phase containing 4 mM sulphuric acid in ultrapure

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water. AsA was detected by absorbance using a diode array detector and quantified at 245 nm.

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Quantification was performed at 245 nm against external calibration of AsA in a range of 20-75

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µg mL-1.

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Sample Extraction, Dilution and Preparation of the Standard Curve for Quantification of

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WSVs

248 249

Extraction procedures followed the protocol from Nurit et al.31 and were as follows: for

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extraction 622 mg (± 2 mg) were weighted into 50 mL tubes (Sarstedt, Germany). Following

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this, 100 µL of internal standard solution (13.6 µg mL-1) was added in addition to 4.75 mL of

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sodium acetate (pH 4.5, concentration of 0.5 mM, pH adjusted with glacial acetic acid), 1.25 mL

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of 0.5 M glyoxylic acid solution, 0.25 mL of 1 % (v/v) L-glutathione reduced solution, 0.25 mL

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of 1 % ethylene-diamine-tetraacetic acid solution (adjusted with NaOH for solubility) and 0.2

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mL of 2 % iron(II) sulphate heptahydrate to the powder. The mixture was strongly mixed for 30

256

seconds using a vortex, and then incubated in the dark at 37 °C for 16 h on a shaker (1,500 rpm).

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Following this, the cooled sample was vortexed and centrifuged at 12,000 g for 10 min at 3 °C.

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The supernatant was filtered through a 0.22 µm filter vial (Polytetrafluoroethylene; PTFE) with a

259

pre-slit cap (Thomson, BioProcess Engineering Services Ltd, Kent, UK).

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Reference materials were prepared to account for extraction, but also instrument stability (see

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Supporting Information; Reference Generation and Quality Control, and Tables S2-S6).

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Chemical Analysis

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Chemical analysis of the potato, field bean and cereal powders were performed on an Agilent

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1260 HPLC system consisting of a quaternary pump, a Diode Array Detector (DAD), a

268

Temperature Control Device, and a solvent Thermostat module (Agilent Infinity 1290) coupled

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to an Agilent 6460A Triple Quadrupole Mass Spectrometer (Agilent Technologies, Santa Clara,

270

CA, USA). Sample extract (5 µL) was injected onto a 100 x 3 mm (2.5 µm) Synergy Hydro-RP

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C18 column with polar end capping, fitted with an AQ C18 4 x 2 mm security guard TM

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cartridge (Phenomenex, Cheshire, UK). Samples were eluted at a flow rate of 0.5 mL min-1

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using a gradient consisting of two mobile phases: A = 0.1 % (v/v) formic acid in deionized water

274

and B = 100 % acetonitrile. The elution gradient was as follows: A/B 98/2 (v/v) hold for 2 min;

275

ramped up from 2 to 60 % B in 3 min and hold for 1 min, and further ramped up from 60 % to 90

276

% in 0.1 min and hold for 1.9 min. Within 0.1 min the gradient was returned to the initial

277

composition and held for 5 min until the next injection. One analytical run lasted 13.1 min

278

(Supplementary Information, Figure S2).

279 280

Mass detection was carried out in positive ion mode for all vitamins apart from the isotopically

281

labelled pantothenic acid (pantothenic acid

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(ESI) interface coupled to the triple quadrupole system. For ESI, the gas temperature, gas flow,

283

nebulizer pressure, sheath gas temperature, sheath gas flow, capillary cap voltage and nozzle

284

voltage were set to 350 °C, 11 L min-1, 50 psi, 300 °C, 11 L min-1, 4 kV (3 kV for negative ion

285

mode) and 500 V, respectively. Collision energies for transition states of the five standard

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compounds including thiamine, nicotinic acid, pyridoxine, pantothenic acid and riboflavin, as

13

C3,

15

N) using a jet stream electrospray ionisation


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well as the isotopically labelled internal standards thiamine-4-methyl-13C-thiazol-5yl13C,

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pyridoxal methyl-d3, pantothenic acid 13C3, 15N, were optimized for optimal fragmentor voltage

289

and collision energies (Supporting [S] Information, Table S1).

290

transitions, i.e. transitions with the highest intensity of the product ions were chosen to build the

291

final multiple reactions monitoring (MRM) method. As shown in Table S1, 16 transitions were

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part of the MRM mode, each with a dwell time of 20 milliseconds (ms) and a delay time of 3.5

293

ms, leading to a total cycle time of 376 ms, and thus 2.7 cycles per second. Peaks of the five B

294

vitamins and three labelled B vitamins were integrated with Agilent MassHunter Quantitative

295

Software (Agilent, USA).

Hereby, the most sensitive

296 297

Quantification of the WSVs

298 299

As listed in Table S1, seven protonated molecular ions [M+H]+ and one deprotonated molecular

300

ion [M-H]- (pantothenic acid-13C3,15N) were chosen as precursor ions in the MS/MS (tandem

301

Mass Spectrometry) experiment. For confirmation, qualifier ions were also included in the

302

method. The most abundant product ions were used for quantification, measured in MRM mode.

303

A ten-point calibration curve for each B vitamin in the potato, field bean and cereal samples

304

were calculated ranging from 2 ng mL-1 up to 1 µg mL-1. As in Nurit et al.31, calibration curve

305

standards were prepared by adding 100 µL of mix unlabelled external standards (10 ng mL-1–5

306

µL min-1) into microfuge tubes containing 100 µL of thiamine-4-methyl-13C-thiazol-5-yl-13C

307

hydrochloride (1 µL min-1), 100 µL pantothenic acid-13C3,15N hemicalcium salt (1 µL min-1),

308

100 µL pyridoxal-methyl-d3 (1 µL min-1) and 100 µL of acetonitrile/water (50:50; v/v). Due to

309

matrix effects, the response ratio of each vitamin was calculated against a labelled internal

310

standard of the same or as similar chemistry as possible: thiamine with thiamine-4-methyl-13C-

311

thiazol-5-yl-13C hydrochloride; riboflavin and pantothenic acid with pantothenic acid-13C3,15N

312

hemicalcium salt and pyridoxine and nicotinic acid with pyridoxal-methyl-d3. 13 ACS Paragon Plus Environment


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Statistical Analysis

315 316

A separate statistical analysis was performed on the vitamin measurements for each crop using a

317

linear mixed model approach. The two main effects of Variety and Input (management system)

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were fitted as fixed effects along with an interaction term. In addition, the effect of Year was

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analyzed as a fixed, rather than random, effect since variance component estimates with so few

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levels can be unreliable. The Years here are considered as experimental replicates and account

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for variability in field and environmental conditions. Three of the terms in the random model

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account for the physical layout of the design. The nested block terms of Replicate (Rep; samples

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taken at the five GPS points, except for field beans, as described below) within Variety strip

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within half-field were fitted as random effects. In addition, interaction terms for Year×Variety,

325

Year×Rep and Year×Input×Rep were also included as random effects. The Year×Variety term

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has been found to be a particularly important component of variance, and necessary for a good

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model fit. The terms for Year×Rep and Year×Input×Rep capture further sources of variance

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within the field. They also account for any variation due to the order in which samples were

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processed in the laboratory stage, as during an analysis run Rep blocks are processed in a

330

sequence with sample order randomised within these (see Supporting Information).

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Between years there were changes to the selection of varieties grown for field beans, Winter

333

wheat, Winter oilseed rape and Winter barley (Table 1). Varieties which were present in only

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one year (Winter wheat – variety Zebedee and Winter barley – variety Flaggon) were excluded

335

from the analysis.

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Data from field beans had a slightly different structure in that the five samples from each strip

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were technical replicates, rather than spatial.

However, the same model was used since


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Year×Rep and Year×Input×Rep terms were still required to capture variability in laboratory

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processing of the technical Rep blocks.

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The complexity of the experimental design meant that there were many potential random effects

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which were difficult to estimate in combination. The model we selected here ensured that the

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fixed effects were estimated against the appropriate level of random variation with degrees of

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freedom estimated from the design. Measured values were logarithmically transformed to base

346

10 (log10) before the analyses to account for variance heterogeneity in the residuals. All analyses

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were performed using Restricted Maximum Likelihood (REML) procedures in GenStat for

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Windows 17th edition (VSN International Ltd., Hemel Hempstead, UK).

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Results

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Five WSVs (nicotinic acid, pyridoxine, thiamine, riboflavin and pantothenic acid; Figure S1)

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were quantified in five varieties of five different crops - potato, Spring barley, Winter barley,

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Winter wheat and field beans. For potato, vitamin C content was also quantified due to its intake

355

being of high importance in western diets41,42.

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In potato, highly significant differences (p < 0.001; thiamine p < 0.01; Table 3) in all six vitamin

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concentrations were seen in all five varieties (Table S8). Most obvious from Table S8 were the

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levels of nicotinic acid, which varied from ~1 – 46 µg g-1 DW, between the varieties (p < 0.001).

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Cabaret had the lowest nicotinic acid levels, whereas the levels in Vales Sovereign were 7-40-

361

fold higher when compared with the other four varieties. Cabaret also had the lowest levels of

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pantothenic acid, riboflavin and thiamine concentrations when compared with the other four

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varieties. Broadly speaking, Vales Sovereign had the highest concentrations of pantothenic acid,



364

pyridoxine and vitamin C. Figure 2 shows that Input did not significantly (Table 3) affect the

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concentrations (Table S8) of any of the six vitamins quantified over the five years studied.

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Table 3 (and Table S9) show significant differences were observed across the field beans

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varieties in the concentrations of nicotinic acid (p < 0.05), pyridoxine (p < 0.01) and riboflavin

369

(p < 0.001). Figure 3 (and Table S9) shows that the variety Ben had the lowest levels of

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nicotinic acid compared to the other seven varieties - Maris Bead, Boxer, Fanfare, Fuego,

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Babylon, Pyramid, and Tattoo. The lowest concentration of pyridoxine was found in Maris

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Bead, whereas this variety had the highest concentrations of riboflavin. With the exception of

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thiamine (Figure 3), whose concentration was significantly lower (p < 0.01) in varieties grown

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under the integrated management system (Table 3), Input did not significantly affect any of the

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other analyzed vitamins (Table S9).

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For Spring barley, with the exception of thiamine (Table S10), highly significant variety

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differences (p < 0.001; Table 3) were observed in the concentrations of four of the WSVs

379

analyzed. As observed for the field beans, Input only affected the concentration of thiamine

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(Figure 4), which again was lower (p < 0.05; Table 3) in varieties grown under the integrated

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management system (Table S10).

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Table 3 shows that for Winter barley all five WSV concentrations had highly significant (p

Impact of Conventional and Integrated Management Systems on the

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