DEGREE PROJECT IN CHEMICAL ENGINEERING, FIRST CYCLE, 15 HP STOCKHOLM, SWEDEN 2020

The role of soil biology to soil and plant health Brandywine tomatoes grown with different microbial additions

MIKAEL ERIKSSON

KTH ROYAL INSTITUTE OF TECHNOLOGY SCHOOL OF ENGINEERING SCIENCES IN CHEMISTRY, BIOTECHNOLOGY AND HEALTH

DEGREE PROJECT Bachelor of Science in Chemical Engineering

Title: The role of soil biology and plant health – Brandywine tomatoes grown with different microbial additions

Swedish title: Jordbiologins roll för jord- och växthälsa – Brandywinetomater kultiverade med olika mikrobiella tillskott

Keywords: Soil biology, soil health, soil food web, inoculation, compost, microbes, tomato plants

Work place: 59degrees

Supervisor work place: Josef Carrey (CEO) and Anette Forsberg (Biodynamic market gardener)

Supervisor at KTH: Gunaratna Kuttuva Rajarao

Student: Mikael Eriksson

Date: 2020-11-17

Examiner: Sara Tyberg Naumann

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Summary

The microbial life in the soil is essential for providing a functioning habitat for plants to grow. A literature study was conducted to investigate the knowledge and science behind soil biology. The purpose of this study was to define what is soil health and how it is influenced by the soil microbial communities. The literature study concluded that the ability of soil biology to benefit plants includes a variety of aspects. Nutrient availability, soil structure and pest resistance are all greatly influenced by soil microbes.

To practically examine these theories, an experiment was conducted where Brandywine tomatoes where grown in three different scenarios. A commercial potting soil, Hasselfors ekojord, was used as substrate in all groups. In the control group (C) the plants were grown only in the substrate. In the second group (R), the seeds where treated with a microbial inoculum and then planted in the substrate. In the third group (RE), the same treatment as in R was done to the seeds and here, compost extract were also added to the RE group. The plants were grown in separate pots in a greenhouse and the growth rate was observed and documented as well as the total harvest. In the end of the growing season a chemical and biological analysis was done to the soil as well as a sap analysis on the leaves. The plant growth where similar among the groups although R and RE showed slightly higher growth rates in the later stages of the growing season. The harvested fruit was highest in C but not significantly. The microbial contents were high in all soils though more fungi communities in the RE and bacterial communities in C. The chemical analysis showed high nitrate concentrations in the leaves in C. In R and especially RE the nitrate conversion into amino acids and proteins where higher wish indicates that these groups are more resilient to pests like aphids.

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Sammanfattning

Det mikrobiella livet i jorden är avgörande för att skapa en fungerande livsmiljö för växter. En litteraturstudie genomfördes för att undersöka nuvarande kunskap och vetenskap bakom markbiologi. Syftet med denna studie var att definiera markhälsa och hur den påverkas av det mikrobiella livet i jorden. Slutsatsen från denna litteraturstudie var att jordbiologins förmåga att gynna växter innefattar en rad olika aspekter. Näringstillgänglighet, markstruktur och skadedjursbeständighet påverkas starkt av jordmikrober.

För att praktiskt granska dessa teorier genomfördes ett experiment där Brandywine-tomater odlades i tre olika scenarier. En kommersiell plantjord, Hasselfors ekojord, användes som huvudsubstrat i alla grupper. I kontrollgruppen (C) odlades växterna endast i substratet. I den andra gruppen (R) behandlades frön med en mikrobiell ympning innan de såddes i substratet. I den tredje gruppen (RE) utfördes samma fröbehandling som i R och kompostextrakt tillsattes också till RE-gruppen. Växterna odlades i separata krukor i ett växthus och tillväxthastigheten observerades och dokumenterades liksom den totala skörden. I slutet av växtsäsongen gjordes en kemisk och biologisk analys av jorden samt en savanalys på bladen. Tillväxten var likartad bland grupperna även om R och RE visade något högre tillväxttakt i de senare stadierna av växtsäsongen. Skördad frukt per planta var högst i C, dock inte signifikant. Den mikrobiella koncentrationen var hög i alla jordar men mer svamporienterat i RE och bakterieorienterat i C. Den kemiska analysen visade högt nitratinnehåll i bladen i C. I R och särskilt i RE var nitratomvandlingen till aminosyror och proteiner högre vilket indikerar att dessa grupper är mer motståndskraftiga mot skadedjur så som bladlöss.

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Content

1 Introduction...... 7 2 Literature background ...... 8 2.1 Soil fundamentals ...... 8 2.1.1 Soil formation ...... 8 2.2 Soil health ...... 10 2.2.1 Soil physics and structure ...... 10 2.2.2 Soil organic matter ...... 12 2.2.3 Soil nutrients and chemistry ...... 13 2.2.4 Soil biology ...... 18 2.3 Compost ...... 26 2.4 Tomatoes ...... 27 3 Plant experiment ...... 29 3.1 Methods and materials ...... 29 3.1.1 Microbial additives ...... 29 3.1.2 Plant experiment setup and cultivation ...... 29 3.2 Observations and analyzes ...... 30 3.2.1 Plant observation ...... 30 3.2.2 Sap analysis ...... 31 3.2.3 Soil analysis ...... 31 3.2.4 Statistical methods ...... 31 4 Results and discussion ...... 32 5 Conclusion ...... 38 5.1 Literature background ...... 37 5.2 Plant experiment ...... 37 References ...... 40

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Acknowledgments

I would like to thank Skillebyholm, Swedish center for biodynamic farming, and their staff for their patience and for providing space in their greenhouses for this experiment. A special thanks to Anette Forsberg for her generosity in both sharing expertise and help.

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1 Introduction

Since the middle of the last century, a gradual use of synthetically produced fertilizers and pesticides as well as the use of fossil fuel as an energy source have increased crop yields. This has provided an increasing population with food, but the optimization of agriculture has often taken place with little regard for long-term effects and has brought with it a number of environmental problems. An obvious effect for us living in Sweden is the bottom death in the Baltic Sea, which is currently spreading on an area as large as Denmark.[1] This can largely be attributed to the leakage of nutrients from agriculture and from other industries. Intensive agricultural methods like heavy tilling of topsoil has also reduced the original carbon rich soil in the US by between 25 and 75%[2]. At the same time, in search for fast-growing crops and high yields has resulted in a steady decline in the nutritional content of cereals, fruits and vegetables.[3]

It is well known that microbial life in the soil interacts with the roots of plants and has a major impact on its growth and health. Optimal biological conditions in the soil can be of great importance in reducing the use of synthetic nutrition and pesticides as well as reducing the need for tillage. At the same time, prosperous soil in the end can also contribute to foods with higher nutritional content. Hence, it is highly essential to have good microbial community in the soil.

This project, assigned by the company 59degrees, will attempt to answer the question of the role of microbial life in the soil to maintain and support the different needs of plants.

A literature study will first answer the questions; What is the basis for the soil as a substrate for plant growth and what are the most important factors for building soil health? What is the microbial life in the soil, “the soil food web”, and what does it contribute to?

An experiment will be conducted where tomato plants will be grown in soil with the addition of different products from 59degrees. Microbial cultures that have been propagated under optimal conditions during composting are added as liquid extract to the soil, solid compost, and by seed inoculation.

The growth and health of the tomato plants was monitored on a regular basis during the growing season and the harvested fruits were weighted. The leaves was analyzed through sap- analysis to give an indication of the nutrient value of the plants. Furthermore, chemical and biological analysis was performed on the soil. All these measures was then used to determine the impact of the added microbial communities to the growth of the tomato plants and fruit yield.

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2 Literature background

This literature background will cover the basic principles behind soil health and especially focus on the biological aspects. Composting principles and basic information on growing tomatoes will also be included since these subjects will be used in the growth experiment.

2.1 Soil fundamentals The soil forms a thin film on the earth’s surface of unconsolidated minerals and organic material and is essential to maintain all ecosystems on which life depends. A well-known and widely cited attempt to define soil is: “the continued capacity of soil to function as a vital living system, within ecosystem and land-use boundaries, to sustain biological productivity, maintain the quality of air and water environments, and promote plant, animal, and human health.”[4]

2.1.1 Soil formation The formation of soil is a complex and dynamic subject as it occurs over time in a wide variety of conditions where climate, geographical conditions and human activity forms soils with different properties. Some general activities of soil formation may however be explained and broken down in three key stages which tend to blend in to each other:[5]

2.1.1.1 Weathering of parent material The soil formation is primary a result of weathering where natural physical, chemical and biological forces breaks down exposed rocks into smaller fractions or into solution.[6] Fragmentation occurs initially due to physical forces such as thermal expansion and extraction, movement and abrasion, pressure from freezing of water and root penetration and swelling. Disintegration increases the surface area and exposes the material to further chemical weathering where minerals are furthered modified through chemical processes such as oxidation and reduction, acidification and alkalization. Some primary minerals are stable and retain their original character in fragmented form while others are more reactive and decompose to form secondary minerals. Prominent among secondary minerals are clay which plays an important role in soil formation. Particles formed due to weathering are not yet true soil but can act as building bricks over centuries or even millennia to form soil. [5]

Depending on the local conditions and the timespan of when soils are formed, the outcome of the soil’s properties will differ. There are five main variables during soil formation that influence the soil characteristics[7]:

- Parent material are the origin of the minerals that form the soil.

- Climate influence affects the rates of dissolution, chemical decomposition, leaching or deposition of the soil components.

- Biotic community influence the soil formation depending on the type of vegetation and types of organisms that are present.

- Topography gives the soil different characteristics due to gravitational effects, for example flat- or slopy-landscape.

- Time is a factor of how long the soil forming process has been active.

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- Human intervention has an increasingly effect on the worlds soil formation due to clearing vegetation, reshaping the land surface and modifying its drainage patterns.

2.1.1.2 Formation of clay and accumulation of organic matter When rock begins to fragment, and form a porous layer containing soluble minerals, water and air will penetrate and plants may colonize the substrate. The residue of living material is later decomposed by microorganisms and soil organic matter will start to accumulate in the soil.[5]

The inorganic particles of soil generally consists of sand, silt and clay particles.[8] Clay particles, less than 2 µm in diameter, plays an important role in building soil structure and fertility mainly due to its extremely high surface area. This surface area contains of negatively charged ions that can bind to nutrients and organic constituents in soil.[9] Clay particles form together with organic material aggregates that are stable and an essential component in building a soil structure.[10]

2.1.1.3 Translocation of matter in different horizons Soils exposed to the force of weathering like water leaching particles, minerals and organic matter will eventually establish a characteristic soil profile. This profile may vary due to different conditions but have been categorized by different horizons normally from top to bottom O, A, E, B, C, R (fig. 1). [11]

The O horizon is the top layer which consists of plant residues ranging from undecomposed to strongly humified organic material.

The A horizon, or surface soil, contains the most accumulated organic material and soil life. Ranging from a few centimeters to a few decimeters in depth. It has a pronounced soil structure. Due to weathering, oxides and clay minerals are formed and accumulated. Sometimes referred to as the zone of elevation due to leaching of soluble components downwards.[12] The eluviation of clay minerals, iron, aluminum, organic compounds etc. can create a Figure 1 Soil horizons [11] lighter colored E horizon at the base of the A horizon.

B horizon, or the subsoil, is described as the zone of illuviation due to minerals and clays leaching from the above A horizon. B horizon is often denser which may inhibit aeration and slow internal drainage.[5]

In the C horizon, or the substratum lies the soils parent material in forms of partially weathered and fragmented rock material. The C horizon devolve into the R horizon , bedrock, which comprise of continuous asses of hard rock that cannot be excavated by hand.[11]

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2.2 Soil health

During the last three decades, there has been a general shift in how we relate to soil. Driven by the organic movements the term ”soil health” has gradually replaced expression ”soil quality”. The underlying principle for the shift is that soil is not an inert, lifeless growing medium but a living ecosystem and only ”living” things can have health. A wide known fact that one tablespoon of soil can contain as many living organisms as the human population on earth. That gives the soil itself the impression of a living organism.[13]

The quality of soil is evaluated using inherent and dynamic soil properties. Inherent soil properties change very little with management, these properties vary with the basic soil formation variables explained earlier; parent material, climate, time etc. The dynamic soil properties on the other hand are management dependent and affected by human management or natural disturbance over a shorter time span.[14]

2.2.1 Soil physics and structure The soil structure is a crucial factor for creating an environment suitable for plant growth. Water availability, nutrient dynamics and soil tilth is greatly influenced by the structure of the soil.

The physical environment of soil is generally characterized by three distinct phases; the solid phase consisting of weathered minerals that forms the soil matrix, the liquid phase comprised of water and the gaseous phase.[8] These inorganic phases together with organic matter can vary proportionally which will influence the soil structure and porosity. An established opinion is that for plant growth, an ideal composition to promote plant growth is 50% pore space equally divided between water and gas, 45 % mineral matter and 5% organic material (fig. 2)[15].[8] Figure 2 Ideal soil distribution for plant growth [15]

The mineral components of soil are sand, silt and clay. There relative proportions determine the soils texture. An overrepresentation of one of these components are in most cases not considered ideal from the perspective of growing plants. In the central part of the textural triangle, with a balanced mix of sand, silt and clay, is a textural class called loam. Loam is considered the optimal soil texture for its capacity to retain water and nutrients and also provides good drainage and aeration. However, for some plant species and in different climates, sandy- or clay- soils may be more suitable than loam.[10]

Apart from the mineral fractions and compositions, which are almost exclusively inherent and influenced by parent material and weathering, an indispensable component for soil structure is humus. Humus is a stable, dark colored fraction of the soil organic carbon and the outcome of

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plant and animal decomposition. These compounds tend to coagulate in association with clay and serves as binding agents for stabilizing, what is called, soil aggregates.[10]

The particles of the soil are often attached together in clusters called aggregates.[10] Soils tend to exhibit an aggregate hierarchy where different hierarchical levels bond together by different mechanisms (fig. 3). These hierarchical structures are what builds the soil matrix to enable porosity and structure for water, gases and plant roots to penetrate. Soil aggregates are generally categorized by microaggregates (<0.25 mm) and macroaggregates (>0.25 mm). Microaggregates forms by clay flocculation together with highly broken down organic material like humus. Carbon stored in the smallest microaggregates are in many cases very stable and can remain undisturbed for hundreds of years. The amount of carbon “sequestered” in soil microaggregates constitutes a substantial fraction of the total amount of carbon on earth. Macroaggregate formation is highly influenced by fine root hairs and fungi. They are generally less stable than microaggregates and can both form and break down much more easily. [16]

Certain basic indicators of how well soils are going to perform physically, holding on to water, providing oxygen to circulate can be measured and observed. Bulk density, is defined by the dry weight of the soil divided by its volume. Medium textured soil with about 50 % pore space will have a bulk density of about 1.33 g/cm3 as unlike rock material with about double that density. Soils rich of organic matter have generally lower bulk density.[17] Slaking is the breakdown of large dry soil aggregates. A slake test can be performed to observe how easily soil aggregates break when they are dropped into water. Rapid water uptake will expose internal stress to the aggregates. If the aggregates break down quickly, they are likely to have poor aggregate stability and vice versa.

Figure 3 Soil aggregate hierarchy [10]

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2.2.2 Soil organic matter The soil organic matter (SOM) is a crucial factor to maintaining soil health. Its presence is a dynamic factor to the soil and therefor highly influenced by how the soil is managed. SOM is a crucial factor for a number of positive properties in the soil. Building soil structure, feeding microbial communities, and keeping nutrients available well established facts of beneficial effects. Soil pH buffering ability[18] and stabilizing soil temperatures[13] can also be added to the list of positive effects of high SOM. If there is no SOM one ends up with a lifeless substrate without ability to support plant growth.

The term SOM includes all the organic compounds in the soil, that is all the living biomass (roots, microorganisms, animals etc.), dead root and plant residues, as well as highly decomposed organic matter no longer identifiable to their origin. The primary source of SOM are residues and debris from plants that fall to the ground and gets incorporated into the soil. SOM can be generally categorized and proportionally divided into fresh residue (<10%), decomposing organic matter (33-50%), stabilized organic matter such as humus (33-50 %) and living organisms (>5%).[13] SOM typically has a water content of approximately 75%. The remaining dry matter consist mostly of carbon, oxygen and hydrogen originally photosynthesized by plant leaves, but also 5% to 10% nutrient elements taken up by the plants from the soil. [13]

The typical molecular compounds in vegetable detritus include:

• Carbohydrates – Ranging from simple sugars to large molecules of cellulose. • Fats – Somewhat more complex molecules than carbohydrates primary found in seeds. • Lignins – Complex compounds found in wood and resistant to decomposition • Proteins – Contains valuable micronutrients such as nitrogen, sulfur. • Charcoal – Derived from incomplete combustion and resistant to decomposition.

2.2.2.1 Decomposition Vegetable detritus in general is not soluble in water and therefor its nutrient compounds is inaccessible to plants. Instead the organic matter is decomposed by microbes which obtain its energy and produce CO2, soil organic matter and soluble nutrients suitable for plant uptake.[19]

In a well aerated soil, all of the organic matter from plant residue will undergo an enzymatic oxidation called soil respiration that in general can be represented as:

푅 − (퐶, 4퐻) + 2푂 → 퐶푂 + 2퐻 푂 + 푒푛푒푟푔푦(478푘퐽/푚표푙 C) 2 푚푖푐푟표푏푖푎푙 2 2 푒푛푧푦푚푎푡푖푐 표푥푖푑푎푡푖표푛 Though there are many intermediate steps as well as side reactions that involves elements other than carbon and hydrogen. One important process is the decay of plant proteins eventually resulting in the soluble inorganic ions such as ammonium, nitrate and sulfate.

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Some of the organic material not lost as carbon dioxide will be used by microbes to synthesize long complex chains that is resistant for further decomposition. These ill-defined complex compounds are called humic substances, or humus.[13]

Estimations say that after one year, approximately two thirds of the organic debris incorporated to the soil oxidizes to CO2. The other portion remains in the soil mostly as humic substances and to some extent as biomass (soil organisms).[16]

There are two determinant factors for the rate of the decomposition of the organic material. One is the composition of the organic compounds, where small organic molecules such as sugars and simple proteins undergo rapid decomposition. Lignins, cellulose and other woody material consisting of long organic molecules will undergo a much slower decomposition.

The other factor for the rate of the decompositions process is the access to nitrogen. Since nitrogen is the main building block for cellular synthesis, an abundance of nitrogen will ensure that microbes can multiply rapidly and therefor speed up the decomposition process. A carbon to nitrogen ratio (C/N ratio) of 25 to 1 is considered an optimal balance to support microbial growth and therefor promotes an optimal decomposition rate.[16]

2.2.3 Soil nutrients and chemistry The functions and properties of soil can be explained physically, biologically, and chemically. These subjects are highly interlinked and are in no way isolated from each other. This chapter will explain some of the main chemical aspects of the soil system.

2.2.3.1 Nutrients For plants to be able to grow and obtain health it is dependent on 18 basic elements, nine in relative large amounts, categorized as macronutrients, and the other nine in smaller quantities, called micronutrients (table 1). Macronutrients Micronutrients Through the process of photosynthesis plants will obtain carbon from the atmosphere and hydrogen and oxygen Carbon (C) Iron (Fe) from water, whereas all the other nutrients are derived from the soil. The presence of these nutrients in the soil Oxygen (O) Manganese (Mn) can be categorized as: Hydrogen (H) Boron (B) • Readily bioavailable – present in the liquid soil solution and available for plant uptake. Nitrogen (N) Zinc (Zn)

• Labile reserve – adsorbed in the soil`s exchange Sulfur (S) Copper (Cu) complex or present in rapidly decomposable organic material. Phsphorous (P) Chlorine (Cl)

• Stable reserve – Present in unweathered minerals Potassium (K) Molybdeum (Mo) or in slowly decomposable organic compounds (humus)[21] Calsium (Ca) Cobalt (Co)

Magnesium (Mg) Nickel (Ni)

Table 1 18 Essential nutrients for plant growth

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Figure 4 Schematic presentation of the nitrogen cycle and the different microbial organisms in various stages of the cycle. [23]

Nitrogen As the most common element in the earth atmosphere, making up 78,1 % of the entire volume, nitrogen is not very abundant in the earth’s crust.[22] Nitrogen is a vital component of proteins, RNA/DNA, and chlorophyll, making it one of the most important elements for plant growth.

The gaseous N2 in the atmosphere is unavailable for most plants and the nitrogen must therefore be available in soluble form. Nitrogen fixing bacteria are a branch of microorganisms that, as the name states, fixes nitrogen directly from the atmosphere (fig. 4)[23]. This activity is the main driving factor in the nitrogen cycle. One group of nitrogen fixing bacteria, known as rhizobium, form symbiosis with certain types of legume plants, including clover, alfalfa, peas and beans. The bacteria attach themselves to the roots as visible nodules and exchanges nitrogen in substitute for sugars.[21]

+ Plants take up nitrogen in inorganic soluble form either as ammonium ions (NH4 ) or nitrate - ions (NO3 ). Though, nitrogen is mostly present in the soil bound to organic molecules, often as amine groups (R-NH2) . These amine groups can be broken down by soil microorganisms into simple amino compounds. Through a vital process called mineralization, the amine groups (-NH2) are hydrolyzed and the nitrogen is released as ammonium ions. Ammonium ions can then be furthered oxidized to form nitrate ions.[21]

Plants respond quickly to increased availability of nitrogen particularly encouraging aboveground vegetative growth and giving the leaves a deep green color. Healthy plant foliage generally contains 2,5 to 4,0 % nitrogen.[16] Plants deficient in nitrogen show a pale green color (chlorosis), develop thin spindly stems, has a low shoot-to-root ratio, and mature quickly (fig. 5)[25]. Nitrogen is quite mobile within the plant and will be transferred to the newest foliage if the plant uptake is inadequate. An oversupply of nitrogen on the other hand can cause excessive vegetative growth and creating top heavy plants. It may also delay maturity and cause the plants to be more 14 (43)

susceptible for disease and to insect pests. Other effects that can appear positive following nitrogen oversupply is large crop yields. Though crop quality often suffer from low sugar and vitamin content as well as poor color and flavor.[16]

Sulfur Sulfur is a constituent of several important amino acids, vitamins and protein enzymes. Closely associated with nitrogen in the process of protein and enzyme synthesis.[13] Sulfur containing compounds are important for the physiology of plants and for resistance to environmental stresses and pests. Sulfur is also involved in plant functions such as photosynthesis and carbon and nitrogen metabolism. [24] Plants absorbs sulfur mainly from soil 2- solution in the form of sulfate anion (SO4 ). Unlike nitrogen, sulfur can also be captured directly from the atmosphere as sulfur dioxide (SO2) or solubilized from parent material such as sulfate minerals.[13] However, the main source of sulfur is derived from soil organic material. Organic sulfur is mostly common in the form of ester sulfates (C-O-S) and carbon bounded sulfur (C-S), and is made plant available through microbial mineralization. Symptoms of sulfur deficiency are similar to Figure 5 Chart of nutrient deficiencies as they appear on leaves. [25] those associated with nitrogen deficiency such as light green or yellow appearance and thin stems.[13] Unlike nitrogen though, sulfur is not mobile within the plant which results in uniformly pale leaves. Under mild deficiency, however, visual symptoms may not always be visible. [24]

Phosphorus Phosphorus is on a basic level an essential element for both animals and plants. This is mainly because it is a constituent of the organic compounds adenosine triphosphate (ATP), often called energy currency of the living cell, as well as DNA and RNA.[13] To be available as nutrients to plants, phosphorus must be present in the soil solution as the 2- - anions H2PO4 or HPO4 . The phosphorus compounds found in soil is mostly highly insoluble, even when soluble sources are added from fertilizers and manure, they are eventually fixed in insoluble form.[21] The total phosphorus level of soils is often relatively low, usually no more than one-tenth to one fourth that of nitrogen. Fortunately, most plants are efficient at utilizing phosphorus. Additionally, most undisturbed natural ecosystems can conserve and circulate this nutrient in its biomass since phosphorus does not form gases that can escape into the atmosphere. Once

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cleared for agricultural use though, phosphorous levels is likely to reduced due to erosion, runoff water and biomass removals.[13] These conditions lay the foundation for intense use of phosphorus as a fertilizer.

Potassium Among essential elements potassium is the third most likely to limit plant productivity, after nitrogen and phosphorus. Potassium is known for activate over 80 cellular enzymes in plants responsible for energy metabolism, starch synthesis and photosynthesis among others.[13] Potassium also acts as an regulator of drought tolerance and water-use efficiency. A deficiency in this element causes chlorosis particularly on the older leaves since potassium is able to relocate to younger leaves in case of deficiency.

Potassium is present in soil in three forms: in organic matter, as exchangeable K+ adsorbed to negative clay particles, or in solution as cation K+. Potassium does not form any gases that can be lost to the atmosphere. Unlike other essential elements potassium availability is not directly influenced by microbiological processes but are influenced primarily by soil cation exchange properties.[21]

Calcium and magnesium Calcium and magnesium are major constituents of minerals in most soils and are among the most abundant cations on exchange complex. They are taken up by plants in large amounts and deficiencies are relatively rear but can occur in acidic sands and leached soils.[21] These nonacid cations can raise pH in acid soils by lowering the levels of cation saturation and enhance pH buffering. Calcium and magnesium are also essential nutrients for plant, animal and microbial life, especially important in helping plants overcome a wide range of environmental stresses.[13]

Micronutrients The elements categorized as micronutrients are all essential elements for plant growth but required in small quantities. That does not imply that these elements are less important, too little or too much of these elements can have dramatic effects on plant growth, low yields, and even plant death.[13] Soil erosion, long-time cropping, increasing crop yields and replacement of nutrient-rich manures with mineral fertilizers have reduced micronutrients in many soils. This has led to a higher awareness of the importance of these elements.[26]

2.2.3.2 Cation exchange capacity Most clay particles and organic matter in soils has negatively charged surfaces and due to their extremely high surface area they have a high potential to adsorb positively charged cations. Many of these cations are plant nutrients such as K+, Mg2+, Ca2+ and NH4+. The loosely bound cations on these surfaces can easily be exchanged by other cations.[10] Plants produce H+ ions that travel to the outer root hairs and can be exchanged for these positively charged nutrients on the clay surface. The soils total ability for this exchange process is called cation exchange capacity (CEC) and is of great importance for the soils ability to store and provide nutrients for plants. CEC can be quantified and expressed in terms of moles positive

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charge adsorbed per mass unit. Organic colloids have a very high CEC which results in soils with high organic matter often have a high ability to store positively charged nutrients. [13] Base-ion saturation is another measure related to CEC though it

2.2.3.3 pH Soil pH is a variable that affects a wide range of soil chemical and biological properties, influencing the like hood that plant roots will take up both nutrient and toxic elements. Soil pH also has a pronounced effect on microbial communities and activity, for example acid conditions inhibiting symbiotic nitrogen-fixing bacteria. Different soil pH affects the nutrient availability of important macronutrients. Nitrogen, phosphorus, potassium and sulfur diminish under acidic and in some cases alkaline soil.[27]

The pH of soil can vary widely, from about two for very acidic soils to nine or more in alkaline soils. Acid soils forms most often in humid regions with high rainfall, this is due to the carbon dioxide in the atmosphere dissolving in water and forms carbonic acid. Water that drains through soil also leaches out base cations such as Ca2+ and Mg2+ increasing the percentage acid-forming cations of H+ and Al+. [28]

Another source of acidification is the respiration of carbon dioxide by soil microorganism, again dissolving in water to form carbonic acid. Plant roots also affect pH by exuding H+ ions into the soil solution in exchange when taking up nutrients from the clay surfaces.

The most predominant effect of soil acidification is aluminum toxicity. Aluminum is present in all soils but soluble in low pH. High concentrations of dissolved Al3- acts toxic to plants by damaging roots and inhibiting root growth. Also, free Al3+ ions can be hydrolyzed and thus, H+ are released and making the soil further acidic. [29]

One important factor to soil pH fluctuation is the amount of clay and soil organic matter in the soil. Clay and soil organic matter helps buffer pH by providing many negatively charged sites and therefore can bind to H+ in acid soil or by releasing H+ in alkaline soil, in both cases pushing soil solution towards neutral. [13]

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2.2.4 Soil biology

All the functions and properties of the soil discussed in earlier chapters are all to a high degree influenced by microbial activity. From the forming of soil, to building soil structure, providing nutrients, and breaking down organic material. Healthy soil is home to enormous diversity of microorganism and animals. A wide variety of organisms exists in the soil ranging from the smallest single-celled bacteria, to animals that can be visible such as ants and earthworms. It is ultimately the life of these microbes, feeding organic material, moving around in the soil, and dies or been eaten, that results in the positive effects on the soil.

2.2.4.1 Diversity Soil host the highest density of microorganisms of any domain in the biosphere, holding more than twice as much as the world’s oceans. It also has the highest diversity.[30] The diversity of different species is highest among the smallest. In a healthy soil, thousands of different species of bacteria and archaea can be found and also 100 species of nematodes while different types of earthworms can be 5-10.

The degree of diversity of different organisms in the soil has been found to be of great importance for maintaining a high degree of functional diversity – the capacity to carry out a wide variety of processes.[13] A high degree of diversity allow the soil to support a diversity of plants as they go through different stages of growth and meet different conditions. In healthy soils there are often many different species capable of carrying out each of the thousands ongoing enzymatical or physical processes. This leads to functional stability that can be resilient and withstand disturbances. Although the activity of a few keystone species such as nitrifying bacteria or burrowing earthworms can influence the health of the entire soil ecosystem. [30]

To illustrate the importance of maintaining a diversity among microbial communities one can for example look at a population of microbial generalist predators. They will be available to deal with a variety of pests but they can only be maintained between pest outbreaks if there is a constant source of non-pest prey to eat. A dilemma in pest control is that pesticide application has enormous effects on non-targeted species in the food web. This together with tillage and monocultures depletes soil diversity and as the diversity declines, new pest outbreaks are much more likely to increase.

2.2.4.2 Nutrient cycles and microbial activity

As mentioned earlier, plants are dependent of 18 essential nutrients where 15 of them is taken up from the soil. When plants or other living organisms dies and falls to the ground, the nutrients tied up in their bodies are not directly available for new plants. It is the soil microorganisms that breaks down these organic bound nutrients to plant available form in a process called mineralization. The soil microbes releases exoenzymes which depolymerize the dead organic matter into simpler molecules. These molecules can then be mineralized by microbial decomposers.[31] When plants absorb the nutrients that has been made available from the decay of dead organic material the circle is closed.

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2.2.4.3 Soil food web The term “soil food web” is often used to describe the complex network in which energy is transferred between different organisms (fig. 6)[32]. The energy primary derives from solar radiation taken up by plants, and is passed upwards through the different levels of the food web.

• Primary producers, typically plants or algae, combine atmospheric carbon with water and energy from the sun to build organic molecules and living tissues. The organic materials contain both carbon and chemical energy that other organisms can utilize. • Primary consumers are organisms that feed on living or dead parts of primary producers. This group can be categorized as herbivores; organisms that eat live plants. Parasitic nematodes, certain termites, woodchucks etc. are herbivores and are also referred to as pests. The vast majority of the primary consumers however, are detritivores, since they feed on plant detritus. • Secondary consumers obtain their energy by consuming primary consumers and can further be categorized into; carnivores – animals that eats other animals, and microbivores – who use microbes as their food source. • The tertiary consumers are at the top of the food chain and consume both primary and secondary consumers. The vast majority of the decomposition of dead plant and animal debris is carried out by saprophytic microorganisms: Bacteria, fungi and archaea. [13]

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Figure 6 Organisms and food chains in the soil food web. [32]

Soil prokaryotes: bacteria and archaea

Bacteria and archaea are prokaryotes which means that their cells lack a membrane surrounding its nucleus. Prokaryotes range in size from 0,5 to 5 m which is similar to a clay particle. Even though bacteria and archaea are similar when observing them under a microscope they are evolutionary quite different.[13] One important property that separates bacteria from archaea is the chemical composition of their cell walls. A teaspoon of soil can contain between 100 million to 1 billion prokaryotes. Under normal conditions this number is dominated by bacteria while archaea are more common in more extreme conditions such as highly acid or alkaline soils, deeply frozen ice or anaerobic sediments. [6]

Most soil prokaryotes, both in occurrence and diversity are heterotopic, living on organic material in the soil. Though certain prokaryotes obtain their energy from sunlight or oxidation of inorganic constitutes, making them autotropic. Heterotopic bacteria along with fungi account for the general breakdown of organic matter in soil. Bacteria often feed on easily decomposed substrates such as sugars, starches and proteins.[13]

Prokaryotes can be seen as social creatures and often acts together in a coordinated manner to form so called “super organisms”, somewhat similar to ant colonies. These colonies can often appear locally where conditions are favorable. Their extreme ability of reproduction enables fast adaptation to changing environmental conditions and food supply.[33] One noticeable effect of this is when gathering organic material and building a compost pile with an optimal carbon to nitrogen ration. Within a short period of time the bacterial populations will grow exponentially and the bacterial activity can make the core of the compost heating up to around 70 °C.[34]

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Soil bacteria beneficial to plant growth, active in the rhizosphere, is referred to as plant growth promoting rhizobacteria (PGPR). Plants can attract and host bacteria that can benefit the plant in different states and conditions. It does so by releasing exudates, simple organic compounds that microbes feed on, and thereby creating a symbiotic relationship . PGPR can support plants by facilitating the uptake of nutrients from the soil, preventing plants from diseases and synthesizing particular compounds for the plant.[6]

There are many ways that bacteria positively influence the soil to both direct and indirect promote plant growth. Some examples are:

- Soil structure – Many bacteria produce a slime- like layer of polysaccharides or glycoproteins that coats the surface of soil particles. These substances play an important role in cementing stable microaggregates that improve soil structure.[33] Another beneficial activity is the humification process that occurs in bacterial metabolism creating organic polymers that are important to soil structure.[13]

- Nitrogen fixation – Nitrogen fixating bacteria, diazotrophs, are responsible for almost all of the nitrogen converted from atmospheric gas to Figure 7 Nitrogen-fixing bacteria colonizing roots and molecule bound nitrogen, such as ammonia. forms nodules. [35] These bacteria often forms symbiotic relationships with legume plants such as peas and alfalfa, producing ammonia in exchange for carbohydrates. Because of this relationship, nitrogen can be incorporated to the soil making it more fertile (fig7)[35].[36]

- Nutrient immobilization - While ingesting organic bound nutrients, prokaryotes immobilizes these compounds in their bodies making these nutrients protected from leaching. Excess amounts of these compounds may be excreted into the soil as inorganic form either by the prokaryotes themselves or by other organisms consuming the prokaryotes.[6]

- Pest control - Beneficial rhizobacteria can protect the plant through a process called induced systemic resistance. In this process the rhizosphere gets colonized by bacteria that cause accumulation of signaling chemicals. These chemicals are translocated up to the plant leaf cells which then forms a defense against a specific pathogen. Induced systematic resistance can occur both before the arrival of a pathogenic pest, as a preventing strategy. The process can also start when an infection has already begun, by the plant root releasing extrudates and thereby attracting bacteria that then stimulates the plants immune system.[13]

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Actinomycetes

Actinomycetes are a class of highly beneficial bacteria that are similar to fungi in such way that it forms long filaments. This branch of bacteria are often among the most abundant of the prokaryotes in soil, especially in soils where acidity is not too great and containing high amounts of organic matter. It has been suggested that some actinomycetes use their branching filaments to connect soil particles so they, along with the soil particles, become too big to be eaten by their natural predators.[6] Unlike many other soil bacteria, actinomycetes are able to break down resistant compounds, such as cellulose and chitin into simpler forms. Because of that they are very important in the final stages of composting when most of the easily metabolized substrates has been used up. Actinomycetes is beneficial to plants by fixing atmospheric nitrogen and help suppress decease. [13]

Fungi

Fungi are eukaryotic, mostly multicellular microorganisms with a filamentous vegetative body. As heterotrophs, they depend on living or dead organic materials for their carbon and energy. Fungi usually grow from spores into thread-like structures called hyphae where liquids can flow relatively freely. The hypha is generally larger than a bacterium, 2-15 m long with a diameter of 0.2-3.5 m.[6] Unlike bacterial cells, fungal hyphae can travel over distances measured in feet or metes which allows them to locate new food sources and transport nutrients from one location to another. Fungi are very effective and versatile in breaking down organic material including cellulose, starch, lignin, as well as easily metabolized compounds. Its ability to tolerate low pH is especially important in decomposing organic residues in acid forest soils. Fungi plays an important role in soil structure as it contributes to the formation of stable aggregate structures. It does so by entangling soil particles to form macroaggregates and by producing glomalin which works as a glue, stable enough to withstand disruption during wetting and drying. [37]

Mycorrhizae

One important activity of soil fungi is the symbiotic relationship between certain fungi and plants. This association is called mycorrhizae and is similar to the plant-bacteria relationship in that the plant secretes simple organic compounds, exudates, that serves as food for the fungi. The fungi then acts as an extension for the plant roots to provide nutrients to the plants from places where root threads cannot penetrate (fig 8)[38].[13] Mycorrhizae fungi are divided into two branches, ectomycorrhiza and endomycorrhiza. Ectomycorrhizal fungi grow close to the surface of plant roots and form webs around it. The other, endomycorrhiza penetrates the roots and grow outwards into the soil.[6] One of the most well-known benefits of the activity of mycorrhizal fungi is its ability to greatly enhance the ability for plants to take up phosphorus from low-phosphorus soils. Mycorrhizae is also known to suppress soil borne diseases by competing with fungal pathogens for infection sites. Mycorrhizae fungi can also form interconnection between plants through their hyphae treads, making it possible for plants to exchange nutrients. For example, a nitrogen-fixing legume can send nitrogen to a nonlegume plant and get phosphorus back in return.[13] 22 (43)

Figure 8 Mycorrhizal fungi stimulating grass growth [38]

Protozoa

Protozoa are single-celled organisms with a nucleus, making them eukaryotic. They measure between 5 to 500 m and are almost always heterotrophs obtaining their nutrients mainly from bacteria, but sometimes fungus and other protozoa.[6] Protozoa are classified into three groups based on their shape. The largest, Ciliates, are the largest and move by waving hair like structures, eating bacteria and other smaller types of protozoa. The Amoebae are smaller and move by forces of extension and contraction. Flagellates are the smallest of protozoa and move by waving whip-like appendages called flagella.[13] Protozoa play an important role in mineralizing nutrients, mainly because of consuming bacteria. The concentration of nitrogen in a protozoa cell is lower than in the bacteria resulting in the protozoa excreting excess nitrogen in the form of ammonium (NH4+). By eating bacteria, protozoa also regulates the bacterial populations by stimulating their growth by the same way as pruning trees can enhance new growth.[39] All bacteria are not available to protozoa though, small pores in soil aggregates can provide shelter for bacteria and bacterial slime can be hard for protozoa to penetrate.[6]

Nematodes

Nematodes are highly mobile unsegmented worms about 4-100 m in diameter and 40-1000 m long making some of them visible by eye (fig. 9)[40]. They are sensitive to water content and porosity since they move in water films in large pore spaces. When soils become to dry nematodes go into a cryptobiotic, or resting state.

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Nematodes can be divided into four broad groups based on their diet. Bacterial-feeders, fungal-feeders, predatory feeders and omnivores. The predatory feeders feed on other nematodes, protozoa and insect larvae. Frugivorous nematodes are known to suppress certain plant parasitic soil fungi and is used as biological control.[41] Some Nematodes can infest the roots by piercing the plant cell with its sharp, spear- like mouth. This activity is very common in plant roots and often has no effect, though high levels of root feeding can result in serious stunting of the plant. [13] Nematodes plays in many ways the same role as protozoa in nutrient mineralization and stimulation on growth on fungal and bacterial populations. Nematodes can significantly influence the distribution of bacteria and fungi through the soil by carrying the Figure 9 Caenorhabditis elegans, a transparent microbes on its surface and in their digestive systems. nematode living in temperate soil. [40] This helps bacteria, which by itself has a very limited mobility, to get access to new food supply.

Arthropods

Somewhere around three-fourths of all living organisms are arthropods. They earned their name from their jointed (arthros) legs (podos) and have no backbone and rely on external exoskeleton.[42] Arthropods are the most species-rich members of all ecological guilds and include springtails, beetles, ants, spiders, mites, centipedes and millipedes to name a few.

Arthropods are enormously significant for nutrient release in the soil and play numerous of roles in the soil food web. They can be categorized in shredders, predators, herbivores or fungivores based on their functions in the soil. [42] Most soil arthropods are shredders that chew on organic matter creating smaller pieces and as they feed, they play an important role in aerating and mixing the soil. This activity is crucial for creating larger surface area for smaller fungi or bacteria to further break down the material. Mites and springtails are alone responsible for breaking down up to 30 % of the leaves deposited on a temperate forest floor.[6] Like nematodes and protozoa, grazing arthropods feed on bacteria and by that stimulating their growth. Much like nematodes, arthropods carries around bacteria on their exoskeleton and through their digestive system and thus mixes bacteria in the soil. [42]

Earthworms

Earthworms are tubular, segmented worms which are hermaphrodites, which means that they carry both male and female sex organs. They are classified into three main categories depending on how they interact with the soil environment. The large anemic earthworms make vertical, relatively permanent burrows, sometimes several meters deep. They effectively burrows surface litter, incorporating it into the soil. Epigeic earthworms are nonburrowing and live in the litter layer very near the soil surface or in composts, hastening the decomposition process. Endogeic earthworms lives mainly in the upper 10-30 cm of mineral soil where they make shallow, horizontal burrows. [13]

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Each time soil passes through an earthworm or arthropod, it is thoroughly mixed with organic matter and mucus and deposited as fecal pellets. Fecal pellets are a highly concentrated nutrient resource, and are a mixture of the organic and inorganic substances required for growth of plants as well as bacteria and fungi. In many soils, aggregates between 0.0025mm and 2.5mm are actually fecal pellets.[42] Compared to the bulk soil, these casts are significantly higher in bacteria, organic matter, and available plant nutrients. When the earthworms die and decay, the nutrients in their bodies are readily released into plant- available form. Studies have shown that where earthworm populations are large, a major proportion of the N taken up by plants (50–90 kg N/ha) can be made available by this mechanism.

One soil health inhibiting factor are the channels created by earthworms. These serve as important pathways for water percolation, aeration and root penetration. Plant roots are therefore doubly favored by this as the channels also contain much available nutrients from the worm castings.

Earthworms extensive physical activity in untilled soils are astonishing, with the capability to move 18 tons of soil per acre in one year.[6] This has thereby earned earthworms the title of “nature’s tillers”. Ironically, extensive agricultural tillage largely reduces earthworm populations and by that reduces this positive effect.[13]

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2.3 Compost

Compost is organic material that has decomposed separated from the site where the material has grown. Unlike the decay of organic matter that is left on top of soil, the compost undergoes a slightly different process. A rapid and controlled composting process generates a substantial amount of heat due to the high activity of microbial organism. This effect rarely occurs on the soil surface when organic material, like leaves that has fallen from a tree, is decomposed.

Composting has been used in agricultural practices at least since the early Roman Empire when piling of organic materials where left to decay and then applied to soil the next growing season.[43]

For the compost process to occur there are four main parameters that has to be of consideration:

Carbon – Carbon rich material are the energy which the microbes feed of. Figure 10 A compost windrow turner aerating steaming Examples are browned leaves, straw, compost.[45] saw dust and paper. Often referred to as “brown” material.

Nitrogen – For the microbes to be able to grow and multiply they need nitrogen. Nitrogen rich material, referred to as the “green” material, are for example kitchen scraps, manure or recently growing green plants.

Oxygen – Microbes use oxygen for oxidizing the carbon chains.

Water – Organisms like bacteria needs a certain amount of water to maintain active.[44]

There are different methods of composting but the most common practice is the thermophilic composting where intense aerobic decomposition is performed within big piles of organic material. In order for this process to work the pile has to be well aerated, often by turning or mixing the material (fig. 10)[45]. The water content must sufficient for the microbes to be active but not too high to create anaerobic conditions. The carbon to nutrient ratio (C/N-ratio) needs to be at around 35.[13] The volume of the compost pile must be big enough to isolate the center of the pile from outside conditions. A minimum of one square meter is often recommended.[6]

The thermophilic compost process can be divided into three phases. During the first brief mesophilic stage, the easily available carbon sources and sugars are consumed causing the temperature to raise gradually to 40 °C. This stage take normally one to two days in a well- balanced compost pile. After this, a thermophilic stage occurs where heat loving thermophilic organism feed on the more complex carbohydrates and cellulose.[13] During this stage the temperature can rise to 65°C or more. Due to these high temperatures, pathogens and weed seeds are killed. As the complex carbohydrates and proteins are broken down and begin to 26 (43)

diminish, the metabolic activity and the temperature starts to decrees. During this final, curing stage, the decay of the most resistant carbon material continues. Mesophilic organisms whose spores has protected them from the heat during the thermophilic stage, reassert themselves and replace the thermophilic organisms. Actinomycetes feed on cellulose, lignin, and chitin creating the earthy smell associated with good compost or soil. Due to lower temperatures, other larger microbes such as nematodes, springtails and earthworms returns and by their activity stimulating the increase of soil-binding fungi and bacteria.[6]

The finished product can be applied to soil to add a number of beneficial effects:

• Compost can act as a slow releasing fertilizer due to relatively stabilized nutrients. An example is the high amounts of living and dead microorganisms that store nutrients within their bodies.[6] • The compost is solely organic material and by adding it to the soil the soil organic carbon raises. A recent long term study from University of California has shown that addition of compost, unlike other investigated agricultural methods, can help soil store carbon even in the deep soil layers.[46] • The compost process involves exponential growth of microbes due to the favorable conditions. When compost is applied to the soil it can act as an inoculation of microorganisms.

2.4 Tomatoes Tomato plants are vines growing as a series of branching stems, with a terminal bud at the tip that does the actual growing. Varieties are generally divided into determinate and indeterminate varieties. Tomatoes originally had an indeterminate plant habit where it continuously grows by producing three nodes between each inflorescence (branch with flower cluster). Determinate varieties, often referred to as bush tomatoes, will eventually terminate the stem with an inflorescence and thereby grow to lower final stage. The time from planting to harvest varies from as little as 45 days to more than 100 days.[47]

Tomato plants prefers warm weather with an optimal air temperature between 18,5-26,5°C. Also soil temperature are important with an optimum between 20-30°C. The tomato plant requires significant and frequent quantities of water. Although excess watering can lead to anaerobic conditions in the root area and infrequent watering can cause blossom end root to the fruit. A mature tomato crop uses 2–3 l of water per plant per day when light levels are high.[48]

Tomato plants grows best on fertile soils and have high requirements for the elements K, Ca, Fe, Mn, and Zn and medium requirements for N, Mg, P, S, B and Cu.[47]

Though tomato plants need most of the essential plant growth nutrients, some are of extra importance:

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Nitrogen –Tomato plants have different absorption rates of nitrogen at various stages of growth. Too much nitrogen in early stages lead to strong vegetative growth and inhibits flower development and fruit set. Later on during flowering and fruit set the nitrogen requirement rapidly increases. Signs of nitrogen deficiency are slow or spindly growth and pale green leaves among older leaves while younger leaves stays green.

Phosphorus – Initially, phosphorus is important for early root growth but later on also promotes vegetative growth and fruit set. Signs of deficiency are red or purple color on the underside of older leaves.

Potassium – Potassium is required for high fruit quality and to regulate growth. As fruit forms, the requirement for potassium is increased, since about 70% of the potassium absorbed moves into the fruit. When lacking, fruiting plants are unproductive and older leaves turning brownish on the edges.

Calcium – A lack of calcium shows up as young leaves curling inwards and lacking color, and is often a problem in acid soils. A symptom of deficiency are also blossom end rot on the tomato fruit.

Magnesium – Deficiency of magnesium is common but rarely results in yield reduction. It is often related to competition to other cations (especially K+) in the soil. Symptoms of low magnesium levels are yellow margins on lower leaves.[49]

With different environment and growth conditions like humidity, light levels, temperatures and nutrient availability, tomato plants might respond in either vegetative or generative growth. Generally, when plants are exposed to stress of any kind, they will respond in a generative manner, focusing on producing fruit. Experienced growers will use these factors to “steer” the plants into an optimum state.[50]

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3 Plant experiment

The plant experiment was conducted to examine the influence of different microbial additives in growing tomatoes. The additives where products made by the company 59degrees. Several observations and analyzes were made during the experiment.

3.1 Methods and materials

3.1.1 Microbial additives The company 59degrees has developed various products that in different ways is intended to enhance the microbial activity to support plant growth. The basis of these additives is a compost made under controlled forms in order to grow a high content and diversity of microorganisms. In this experiment a seed inoculation (seed rub) based on this compost was used prior to sowing to introduce microbes to the seed. Thereby beneficial microorganisms was made present in the rhizosphere from an early stage.

A compost extract was also used in the experiment. This extract was prepared by filling fine mesh bags with compost and letting them infuse in water for 20 minutes. Thereby, soil microbes from the dry compost could be released into the solution. This extract was then added to the soil.

3.1.2 Plant experiment setup and cultivation In this experiment three groups where set up with eight tomato plants in each group. The tomato variety was Brandy wine, which is an indeterminant species with large potato-leaved foliage and bears large beefsteak-shaped fruit. The plants where germinated and grown in individual pots to isolate the soil conditions from any outside influence. During the experiment, the plants where transplanted into larger pots as they grew larger. This was done five times during the experiment and the plants finally ended up in 30 liter growbags.

The three groups were named control, seed-rub and seed-rub + extract. The substrate and seed treatment for each group was as following (fig. 11):

Control

Eight plants were grown from seed in KRAV certified potting soil - “Hasselfors ekojord”. No treatment was made to the seed.

Seed-rub

Eight plants were grown from seed in ¾ KRAV certified potting soil - “Hasselfors ekojord” and ¼ compost made by 59degrees. The seeds were inoculated with seed-rub made by 59degrees.

Seed-rub + extract

Eight plants were grown with the same substrate and seed-rub as the previous group. This group did also receive compost extract once a week during the experiment.

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Figure 11 Schematic illustration of the plant experiment The same amount of water was added to each plant manually throughout the experiment.

During the period of harvest, fertilization was added in the form of chicken manure. The fertilization was estimated to provide a dose of 1 gram of nitrogen per week based on feralization recommendations.[49][51] 50 grams of chicken manure was added at day 123 and another dose at day 144.

The plants were pruned regularly, removing side-shoots as they appeared. Damaged fruit, especially fruit that developed blossom end rot (BER) were removed and weighed.

Tomatoes were harvested and weighted when ripen. Damaged fruit, especially fruit that developed blossom end rot where removed and weighed.

3.2 Observations and analyzes Several observations and analysis was done during the experiment.

3.2.1 Plant observation Plant growth and development was observed continuously with these different parameters: • Germination percentage [%]. Observed once every day until first shoot was visible. • Shoot height [cm]. The shoot was measured from the soil level to the top shoot once every 7-14 days. • Amount of leaf branches. The total number of leaf branches where counted once every 7-14 days. • Stem width [mm]. The stem diameter was measured near the soil level once every 7- 14 days. • Shoot weight [g]. The shoots were removed and weighted in the end of the experiment. • Root weight [g]. The roots were drenched in water to remove soil and other particles and then dried for 24 hours in a warm boiler room. After that the roots were weighted. • Overall observation of the plant growth was done once every 7-14 days to look for deficiencies, diseases or other visible signs.

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3.2.2 Sap analysis At the end of the plant experiment (day 155) a sap analysis was conducted. This was done to determine the current nutrient content in the sap fluid. From each plant, two young but fully developed leaves (three and four branches from the top shoot) was collected as well as two old but still vital leaves (8 and 9 branches from top shoot). The young and old leaves in each group were gathered in airtight plastic bags to a total of six samples and sent for sap analysis. A general value of the sap nutrient content within each group was determined. The analysis was done by Nova Crop Control in the Netherlands. Since some nutrients such as nitrate, potassium an magnesium are mobile within the plant, the content of these nutrients might move from the old to the new leaves. If there is a big difference one can suspect that there is a deficiency in the soil and the plant is using its own supply.

3.2.3 Soil analysis Within each group, a similar amount of soil from each plant was collected and mixed together. These soil samples were considered to be a general picture of the soil properties within each group. The soil samples were then analyzed as follows:

• Chemical analysis [Base-ion saturation, pH, Micro/macro nutrients, %Soil organic matter] done by Eurofins Agro Testing Sweden. • Biological analysis [bacteria/fungal/actinomytes biomass and total count of micro arthropods protozoa and nematodes] done by Octavia Hopwood of 59degrees.

3.2.4 Statistical methods Raw data from the observations in the plant experiment was stored in MS Excel. T-test and standard deviation was used to statistically analyze the data.

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4 Results and discussion

Plant growth

After sowing, all seeds germinated in all three groups. Seedlings appeared after four days in the control (C) group, after five days in the seed rub (R) group, and after six to seven days in the seed rub + compost extract (RE) group. This might be explained to temperature differences in the different seed trays due to their placement.

Figure 12 The tomato plants at different stages of growth. From top left to bottom right; day 20, 54, 85 and 121.

The development of shoot growth, stem width, and development of leaf nodes was normal in all groups even though some plants exhibited tendencies to vegetative growth while others leaned to degenerative growth, producing fruit early (table 2). One plant in the R group and one plant in the RE group accidentally lost their top shoot during the experiment and was therefore removed from the experiment. Totally 8 plants were used for analyzing shoot growth, height, weight and leaf nodes.

A t-test was conducted for every observation during the growth experiment. At day 38, the C group had significantly higher (P  0,05) shoot height than both group R and RE. At day 85, group R had significantly higher shoot than group C. At day 78 both R and RE had significantly more leaf branches than C. At the later stages, from day 105 onwards, the

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development of shoot height were slightly higher in R compared to C and RE. A similar tendency was observed with RE which had a slightly higher production of new leaf nodes. The difficulties in consistent measuring might probably explain why stem width goes back in the later stages of the experiment.

Shoot height Stem width 300 17

250 15

) )

m 13

200 m

c

(

m

(

t

h

h

t

g

i

d i

e 150 11

h

w

t

o

m

o

e t

h 100 9

S S

50 7

0 5 0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180 Days Days

C R ER C R ER

Leaf nodes 40 Table 2 Growth rate of tomato plants from three groups on 35

) different cultivation days. An average value from 8 plants t

n 30 u

o were shown. Arithmetic mean line and standard deviation

c

l 25 a

t bars.

o

t (

20

s

e d

o 15

n

f

a 10

e L 5 0 0 20 40 60 80 100 120 140 160 180 Days

C R ER

The weighing of the shoots and roots was problematic and the results were not considered to be reliable. Organic material and larger particles could not effectively be removed from the roots without removing significant amounts from the root mass. The results were therefor excluded.

Fruit yield

The plants started to develop flowers nine weeks after sowing. There were no significant difference in flower development between the groups. The first fruit development occurred at week 11. At day 94 the fruits weight was estimated visually without harvesting. These rough estimations showed no significant differences in fruit production among the groups. During the observations at day 94 and 106 some fruit had developed blossom end rot and was therefore harvested and weighted. There were no significant difference of the weight of the BER fruit between the groups.

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Although the C group had the highest average fruit yield per plant, the fruit yield showed no significant difference among the groups (table 3).

Plant nr fruit yield Plant nr fruit yield Plant nr fruit yield (g) (g) (g) C1 2820 R1 2670 RE1 2540

C2 3930 R2 3800 RE2 excluded C3 2710 R3 2300 RE3 1870 C4 2350 R4 2780 RE4 1600

C5 4600 R5 2300 RE5 3430 C6 2950 R6 3750 RE6 2800

C7 4030 R7 3230 RE7 3220 C8 1610 R8 excluded RE8 4250

Tot (g) 25000 20830 19710

Average 3125 2976 2816 per plant (g) P-value 0,737 0,542

Table 3 Total fruit yield of each plant. P-value from t-test comparing R and RE with Control.

Chemical analysis

The pH of the soils and leaves were similar but slightly higher in RE and the young leaves correlates well with the soil pH (table 4). The higher amount of fungi found in RE compared to C and R would be expected to lower the pH, in this case the effect was the opposite. The lower soil organic matter (SOM) in the RE group could be related to the higher amounts of fungi in this group. Fungi are often more effective in breaking down complex organic molecules than bacterias.

Cation-exchange capacity was allso slightly lower in RE, this might be relatet to the lower amount of SOM compared to C and R.

pH Cation-exchange capacity Soil organic matter (SOM) 7 90 45 6,1 6,2 6,2 5,8 5,8 5,8 80 40 6 5,7 5,3 5 70 35 5

w 60 30

d

4 g

0 50 25

0

% 1

3 / 40 20

q e

m 30 15 2 20 10 1 10 5 0 0 0 C R RE C R RE C R RE soil young leaves old leaves

Table 4 From left to right: pH in leaves and soil, cation-exchange capacity in soil and soil organic matter in.

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The overall macro- and micro- nutrient content of both soils and leaves where simmilar between the groups with some exceptions (table 6 and 7).

The form of nitrogen in the leaves differed amoung the groups. RE had low concentrations of nitrate ions in both the soil and in the sap compared to C (table 5). A high conversion of nitrates to amino acids and proteins is a sign for good resistanse against pests like aphids.[52] The higher conversion of nitrates in RE might be influenced by microbial life. Also since the other limiting factor for nitrate conversion, the availability of magnesium, was simmilar amoung all groups.

Ammonium content in all soils were high. This could lead to ammonium toxicity in the plants. Ammonium toxicity generally appear with external ammonium concentrations above 2 to 9 ppm. Slightly higher ammonium concentration in C compared with R and RE might be rellated to the lower pH in C. Ammonium rich soils are often low in pH. [53]

NH4 Ammonium NO3 Nitrate 600 800 500 700 400 600

500

m m

p 300

p 400

p p 200 300 200 100 11 8,4 8,7 100 0 0 C R RE C R RE soil young leaves old leaves

Total nitrogen (N/A on soil) Amount of total N in NO3 2500 40 2000 30 1500 20 1000 500 10 0 0 C R RE C R RE

young leaves old leaves young leaves %

Table 5 Nitrogen content in soil and leaves.

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Phosphorus P Potassium K CalciumCalsium Ca Ca 800 5000 4000

4000 600 3000

3000

m

m m

p p

p 400 2000

p p p 2000 200 1000 1000 42 76 62 0 0 0 C R RE C R RE C R RE

Magnesium Mg Sulfur S (N/A on soil) 1000 2500 800 2000 soil

600 1500

m

m p

p young leaves p p 400 1000 old leaves 500 200 0 0 C R RE C R RE

Table 6 Macronutrients in leaves and soil Manganese Mn Zink Zn 50 60

40 50 40

30 m

p

m p p 30 p 20 20 10 10 0 0 C R RE C R RE

Iron Fe Sodium Na (soil n/a) 3 600 2,5 500 2 400

soil

m

m p

1,5 p 300 p p young leaves 1 200 old leaves 0,5 100 0 0 C R RE C R RE

Table 7 Micronutrients in leaves and soil

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Microbial analysis

The microbial analysis showed that all soils were healthy in regard to microbial activity (table 8). There were very good amounts of nematodes in all three of the samples and protozoa numbers were generally good. What stood out was much higher amounts of fungi in the RE compared to C and R.

Units Optimal C R RE Total Bacteria μg/g is it 300-1000 1223 1092 1043 dry weight or wet weight? Total fungi μg/g 100-300 326 305 887 hyphal μm > 2.5 4,4 3,8 4,6 diameter oomycetes μg/g <300 0 0 0

Fungi/bacteria ratio 1-5 0,27 0,28 0,85

Nematodes total beneficial no./g 0-5 3 4 3 total root no./g 0 0 0 0 feeding Protoza flagatelles no./g > 10,000 244560 61140 40760 amobae no./g > 10,000 40760 40760 61140 ciliates no./g 0-7000 0 0 0 Table 8 Microbial content in soils

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5 Conclusion

This report attempts to investigate both in a theoretical and practical way the role of biology to soil and plant health. Therefore, the conclusions will be divided between the theoretical literature background and the practical plant experiment.

When going through literature and research related to soil science, one can state that the microbiology plays an essential role in building a functional and healthy soil. The benefits of a divers and healthy microbiota is already an obvious part of the regenerative agriculture movement and one can hope that, going forward, the large scale agriculture will change their practices for maintaining a rich soil life. The microbiology in the soil can help plants in numerous ways, where some of the most essential are:

• Providing nutrients by mineralizing organic bound nutrients, transporting nutrients through fungal hyphae from places where roots cannot reach, and by immobilizing nutrients and thereby protecting them from leaching. • Building soil structure by creating humic substances, releasing slime like glues that binds soil particles into soil aggregates, and by creating channels for water percolation and aeration. • Protecting plants against pests and diseases by stimulating plants immune system Some of the most occurring activities in the modern agriculture are related to the objectives mentioned above. Some of the most important suggested changes in agricultural practices to benefit the microbial life in the soil are:

• Reducing tillage in order not to disturb microbial activity. • Maintaining a high organic content in the soil. The organic content in the soil is what feeds the microbes. One very effective way of building and maintaining soil organic matter is by keeping the soil covered with living or dead material. • Reducing the use of pesticides since they not only kill pests, but also beneficial microbes. The main observed differences in the plant growth between the three groups where that the R group showed slightly stronger shoot growth in the second half of the experiment and that RE had a stronger tendency for producing new leaf nodes. The seed inoculation and added microbiology from compost extract might play a role in this. Earlier research and trials has shown positive results in plant growth and health by inoculating beneficial microbes to seeds and soil.[54][55] Though, repeated attempts of this experiment over several growing seasons must be done to statistically ensure these trends.

The control group had the highest amount of harvested fruit per plant. However, this result could not be statistically distinguished from random factors.

The chemical analysis showed interesting differences in the nitrogen content in both the soil and in the leaves. The RE group showed a much lower nitrate content in the leaves and also a higher rate of nitrate conversion to organic molecules such as amino acids and proteins. The lower nitrate content in RE compared to the control group is likely to make the RE group less

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attractive to sap drinking aphids. The conversion rate of nitrates are highly affected by microbial activity.

The biological analysis of the soils showed that all soils had satisfying amounts of soil microbiology. The main differences were that RE contained much higher amounts of fungi.

The differences in microbial content between the two biological treated groups, RE and R, and the control where smaller than expected. This is probably one explanation why there where such small differences in how the plants performed. On the other hand, the fungal population was approximately 2fold higher in RE soil compared to R and C which might influence the breakdown of complex organic matter.

In this experiment, an expensive, high quality potting soil was chosen as the main substrate. Further experiments of this kind could be set up in such way that the soil, prior to biological treatments, has a poor biological condition. For example, by using soil from treated conventional growing operation where tillage and chemical pesticides has been used.

Recommendations for future work could also be to add more compost extract and seed-rub in order to see more differences.

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