Unravelling the bruising-discoloration of Agaricus bisporus, the button mushroom Amrah Weijn Thesis committee Promotor Prof. dr. H.J. Wichers Professor of Immunomodulation by Food Wageningen University Co-promotor Dr. J.J. Mes Senior Scientist at Food & Biobased Research Wageningen University Other members Prof. dr. W.J.H. van Berkel, Wageningen University Dr. M.P. Wach, Sylvan Research, Kittanning, USA Prof. dr. H.A.B. Wösten, Utrecht University Prof. dr. ing. E.J. Woltering, Wageningen University This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Unravelling the bruising-discoloration of Agaricus bisporus, the button mushroom Amrah Weijn Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 14 June 2013 at 1.30 p.m. in the Aula. Amrah Weijn Unravelling the bruising-discoloration of Agaricus bisporus, the button mushroom PhD Thesis, Wageningen University, Wageningen, NL (2013) With references, with summaries in English and Dutch ISBN 978-94-6173-563-8 Contents Chapter 1: General introduction 1 Chapter 2: A new method to apply and quantify bruising- 37 sensitivity of button mushrooms Chapter 3: Bruising-sensitivity of button mushrooms 59 Chapter 4: Melanin biosynthesis pathway in Agaricus 75 bisporus mushrooms Chapter 5: Phenolic compounds of the melanin biosynthesis 109 pathway in bruising-tolerant and bruising- sensitive Agaricus bisporus strains Chapter 6: A molecular approach to identify key 143 components involved in the bruising-tolerance of the button mushroom Agaricus bisporus Chapter 7: Molecular and biochemical analysis of bruising- 177 tolerance based on Agaricus bisporus strains selected from a segregating population Chapter 8: General discussion 209 Summaries Summary 239 Samenvatting 243 Dankwoord 249 About the author 255 Curriculum Vitae 256 List of publications 257 Overview of completed training activities 259 Chapter 1 Chapter 1 Chapter 1 General Introduction 1 Chapter 1 1 Introduction The Netherlands are the fourth producer of the white button mushroom over Chapter 1 the world (Ministerie van Landbouw, 2010). China is the largest producer of mushrooms with an average production of approximately 2.7 million ton per year from 1992 until 2010, followed by the USA (0.37 million ton) and the Netherlands Chapter 1 (0.24 million ton) (mushroom and truffles, http://faostat3.fao.org/home/index.html #VISUALIZE). In 2012 there were 131 mushroom producing companies in the Netherlands, this is 70 % less then in 2000 (546 companies) (http://www.cbs.nl/nl- NL/menu/themas/landbouw/publicaties/artikelen/archief/2012/2012-champignons- 2012-art.htm). Also the cultivation area decreased since 2000 with 30 % to 665,000 m2 in 2012. Due to production of mushrooms in European countries with lower labour costs the mushroom market in the Netherlands is challenged. In 2010 it was calculated that the production costs for a mushroom grower for the fresh market were 1.31 euro per kg mushrooms in the Netherlands, in the United Kingdom this was 1.14 euro per kg and in Poland 0.97 euro per kg (Visie Nederlandse Champignon Versmarkt, 2010). Labour costs, which are part of the production costs, can be lowered by automatic harvesting. A full automation of harvest is technologically feasible but requires adaptation of the present production system. Next to this, the critical success factor is the availability of strains that are less sensitive to mechanical damage. Contact-based discoloration, or bruising, is caused by a mechanical process known as „slip-shear‟ (Burton, 2004), a downwards force and a sideways movement. This bruising based discoloration of mushrooms can occur during picking by hand or by robotic picking equipment and leads to lowering of quality. Mechanical harvesting is more cost-efficient than picking by hand, but cannot be applied yet to serve the fresh market, as commercial strains are too sensitive to bruising. When strains with less sensitivity to bruising are available an increase in quality for both the fresh and the preserved mushroom industry is possible. 2 Chapter 1 2 Button mushroom cultivation and harvesting The button mushroom Agaricus bisporus is grown indoors in large tunnels. Six Chapter 1 steps are involved in mushroom growth (Royse and Beelman, n.d.). The first step is the production of mushroom compost which lasts around 6-14 days. The substrate for the cultivation of mushrooms is horse manure compost, which consists of a mixture of horse manure, some broiler chicken manure, and water, to which gypsum is added for structural stability and for stabilizing the pH. Alternatively, synthetic compost can be made which is based on wheat straw and broiler chicken manure (van Griensven and van Roestel, 2004). The second step of composting is pasteurization to kill any insects, nematodes, pest fungi, or other pests that may be present in the compost. At the same time it is necessary to condition the compost and remove the ammonia that was formed during the first step. The third step in mushroom growing is mixing the compost with spawn. Spawn consists of hydrated and sterilized grains colonized with a pure culture of mushroom mycelium. Mycelium is thin thread-like cells which can grow vegetatively from germinated spores. It requires around 13-20 days to obtain fully colonized compost. At step four, a top soil layer, called casing, is applied to the spawn-run compost. On top of the casing layer, mushrooms will form. Step five of mushroom growing is the pinning phase, in which tiny mushrooms are formed starting from a pin. The sixth step is called cropping or harvesting. Mushrooms are harvested over a 2-4 day period in a 7-10 day cycle called flushes or breaks (Beyer, 2003). The complete life cycle of A. bisporus was unravelled in the early seventies of the previous century (Figure 1) (Sonnenberg et al., 2011). After an apparently normal meiosis, predominantly bisporic basidia are produced, each containing two non-sister post meiotic nuclei. Upon germination these spores generate heterokaryotic mycelia. Only spores from the rare four spored basidia contain one haploid nucleus and can be used to generate hybrids in breeding programs. This 3 Chapter 1 four-spored variety was found in the Sonoran desert of California and is named Agaricus bisporus var. burnettii (Callac et al., 1993). Chapter 1 Button mushrooms consist of different tissue types, which develop during the growth of the mushroom (Figure 2 and 3). The outer layer of the cap is the skin, which covers the flesh. The gills contain the spores and are protected by the velum in early developmental stages. The mushroom cap is formed on a stem, which is connected via the mycelium to the compost. Figure 3 shows the different developmental stages of the button mushroom defined from stage 1 until stage 7 (Hammond and Nichols, 1975; Hammond and Nichols, 1976). The button mushroom is usually harvested for the market at developmental stage 2 or 3, while the larger Portobello‟s are harvested at a more mature stage. Figure 1. Life cycle of Agaricus bisporus Retrieved from Sonnenberg et al. (2011). 4 Chapter 1 Skin Flesh Chapter 1 Gills Velum Stem Figure 2. Tissues of button mushroom Agaricus bisporus. Adapted from van Leeuwen and Wichers (1999). Figure 3. Development stages of button mushroom Adapted from Hammond and Nichols (1975; 1976). Stage 1: pinhead, is characterized by undifferentiated velum (diameter of the cap is < 5 mm). Stage 2: button, is characterized by visible and intact but not stretched velum (diameter of the cap is 20-30 mm). Stage 3: closed cup, the velum is stretched but still intact (diameter is 30-40 mm). Stage 4: cup, velum starting to tear (diameter 30-40 mm). Stage 5: cup, velum torn, cap still cup shaped, gills clearly visible (diameter 30-50 mm). Stage 6: flat, gill surface flat or slightly concave (diameter is 40-60 mm). Stage 7: flat, the gill surface curving upwards (diameter is 50-70 mm). 5 Chapter 1 3 Mushroom bruising and discoloration 3.1 Methods to analyse discoloration and bruising-sensitivity Chapter 1 Discoloration can be measured with a chromameter, which is a tool for precise and objective assessment of surface colour (Muizzuddin et al., 1990). The chromameter colour measurements are given in the CIE L*a*b* colour space (http://www.hunterlab.com/appnotes/an07_96a.pdf). L* stands for lightness, which is measured from 0 (black) to 100 (white). Positive a* values stand for red colour, negative a* values for green. Positive b* values stand for yellow colour, negative b* values for blue. Several studies used a mechanical shaker to mimic mechanical damage. Burton and Noble (1993) used a polystyrene shaking box which oscillated horizontally to bruise mushrooms. Bruising of mushrooms was caused largely by rolling along the base of the box, but also by collisions with the sides of the box or with other mushrooms. The bruising treatment applied caused discoloration of the mushrooms which was equivalent to approximately seven days of storage at 5 °C or two days of storage at 18 °C without bruising. Noble et al. (1997) used the same technique as Burton and Noble (1993) to bruise mushrooms and found that the sides of the mushrooms showed a higher degree of discoloration than on the top of the mushroom cap. A Gyrotory G2 shaker (New Brunswick Scientifiuc Co. USA) was used at 300 rpm to damage mushroom during a controlled period of time by Esquerre et al. (2009), Gaston et al. (2010) and O‟Gorman et al. (2010). Gaston et al.