grass-clover silage as cut-and-carry fertilizer in organic

Grass-cloversilageascut-and-carry

fertilizerinorganicpotatoproduction:

InfluenceofC:Nratioandnitrogenapplicationrateofgrass-

cloversilageoncropgrowthandyield

MScthesisreport

Pauline Martel

MSc Organic Agriculture

Supervisor: Egbert Lantinga

Farming Systems Ecology Group

Wageningen University, Wageningen

The Netherlands

Pauline Martel MSc Thesis Report

2

Grass-clover silage as cut-and-carry fertilizer in organic

potato production:

Influence of C:N ratio and nitrogen application rate of

grass-clover silage on crop growth and yield

Pauline Martel

March 2016

Registration number: 930220545020

MSc Organic Agriculture

Course code: FSE-80436

Supervisor: Egbert Lantinga

Examiner: Jeroen Groot

Farming Systems Ecology Group

Wageningen University, Wageningen

The Netherlands


3

Table of contents

Acknowledgements ............................................................................................................................. 5

Abstract ............................................................................................................................................... 6

Abbreviations ...................................................................................................................................... 7

I- Introduction ................................................................................................................................. 8

1) Background and Rationale ...................................................................................................... 8

2) Diagram of the research design ............................................................................................ 10

3) Purpose of the study and Research questions ...................................................................... 11

II- Material and methods ............................................................................................................... 12

1) Experimental site: Droevendaal farm.................................................................................... 12

2) Experimental design and management ................................................................................. 12

3) Preliminary experiments ....................................................................................................... 14

4) Field measurements and laboratory analysis ........................................................................ 16

5) Statistical analysis .................................................................................................................. 19

III- Results and discussion ........................................................................................................... 20

1) Conditions during growing season ........................................................................................ 20

2) Plant growth: emergence date, plant height, canopy volume, light interception ................ 20

3) Yield components: fresh yield, dry matter yield, agronomic efficiency and tuber N

accumulation ................................................................................................................................. 25

4) Nitrogen dynamics: tuber apparent N recovery and N released from silage ....................... 29

IV- Conclusions ............................................................................................................................ 32

V- References ................................................................................................................................. 33

VI- Appendices ............................................................................................................................ 37


4

Table of figures

Figure 1. Diagram of the research design ............................................................................................. 11

Figure 2. Cumulative weekly precipitations and minimum (Tmin) and maximum (Tmax) averaged

weekly temperatures during potato growing cycle (22/04/2015-10/09/2015). .................................. 12

Figure 3. Experimental layout ............................................................................................................... 13

Figure 4. Plant height at 7 (a), 9 (b) and 11 (c) weeks after planting as affected by C:N ratio. ............ 21

Figure 5. Plant height at 7 (a), 9 (b) and 11 (c) weeks after planting as affected by N fertilization rate.

............................................................................................................................................................... 21

Figure 6. Canopy volume at 7 (a), 9 (b) and 11 (c) weeks after planting as affected by C:N ratio. ....... 22

Figure 7. Canopy volume at 7 (a), 9 (b) and 11 (c) weeks after planting as affected by N fertilization

rate. ....................................................................................................................................................... 22

Figure 8. Light interception over time as affected by treatments combining C:N ratio and N

fertilization rate. .................................................................................................................................... 25

Figure 9. Fresh tuber yield as affected by C:N ratio (a) and N fertilization rate (b). ............................. 26

Figure 10. Fresh tuber yield as affected by combined treatments of C:N ratio and fertilization rate (kg

N ha-1). ................................................................................................................................................... 26

Figure 11. Agronomic nitrogen efficiency as affected by C:N ratio. ..................................................... 27

Figure 12. Tuber nitrogen accumulation as affected by C:N ratio. ....................................................... 28

Figure 13. Tuber apparent nitrogen recovery as affected by C:N ratio (a) and N fertilization rate (b). 29

Figure 14. Tuber apparent N recovery as affected by combined treatments of C:N ratio and

fertilization rate. .................................................................................................................................... 30

Figure 15. Nitrogen release over time (in weeks after burying) from decomposition of silage in

litterbag experiment of C:N ratio 16; 17; 22; 24. .................................................................................. 31


5

Acknowledgements

This study was conducted at the Farming Systems Ecology group as part of my first MSc thesis, which

is one of the fundamental parts of my double-degree in “Agroecology” between Wageningen

University, the Netherlands and ISARA-Lyon, France.

The completion of this research was made possible thanks to the contribution of some people.

First of all, I thank my supervisor Egbert Lantinga who guided me through the whole study and

Johannes Scholberg for his precious advice.

I am thankful to my colleagues Ivan Palomba and Claude Majuga for their collaboration.

I would like to thank Andries Siepel, John van der Lippe, Wim van der Slikke and the rest of the staff

from Unifarm for their contribution and help in the elaboration of the field experiments; Hennie

Halm for the laboratory analysis and Peter van den Putten for his technical support.

Special thanks to Pierre, Thibault and Harimurti for their help in statistics.

Last but not least, I am deeply thankful to my family and friends for their assistance, compassion,

patience and support.


6

Abstract

More and more organic farmers are using cattle manure or slurry as their major source of nutrients.

However, the availability is limited already for organic operations, and regulations are becoming

increasingly strict concerning the use of non-organic animal-based manure. An innovative agronomic

practice in Dutch organic agriculture consists in applying cut-and-carry fertilizers, which is plant material

with a high nitrogen content that is being harvested and stored before being applied later to other fields.

Grass-clover silage can be one of them. However, previous applications of grass-clover silage with a C:N

ratio higher than 20 resulted in nitrogen immobilization during the first weeks. The objective of this

study was to investigate the effect of C:N ratio (16; 17; 22; 24) and nitrogen application rate (0; 57;

113; 170 kg N ha-1) on potato growth and yield. Results showed that use of C:N ratio above 20

resulted in lower growth performance in the early stage of canopy growth (9 weeks after planting)

and lower potato yield, although there was a yield difference of 10% only with C:N ratio 16. Nitrogen

accumulation in the tubers was not influenced by C:N ratio, but there was somewhat a decrease in

the apparent nitrogen recovered in the tubers as the C:N ratio increased. Regarding nitrogen

application rate, plant height and canopy volume increased with increasing rates at a later stage of

canopy growth (11 weeks after planting). There was a positive linear trend with fresh potato yield

and with nitrogen accumulated in the tubers. Also, nitrogen recovered in the tubers somewhat

increased with increasing fertilization rates. Based on these results, grass-clover silage can be

recommended as a suitable alternative to cattle manure. Special attention should be given to

nitrogen application rate, from both economic and environmental approaches. A subsidiary

experiment with litterbags to follow the nitrogen release pattern from silage showed there was no

clear effect of C:N ratio. However, only the release of total nitrogen was taken into consideration, so

that further research may be done regarding nutrient release patterns of plant material with varying

C:N ratio. Also, unexpected results were found regarding effect of fertilization rate on yield

components, including strict linear trend between fertilization rate and fresh tuber yield, and

increasing values of apparent nitrogen recovered in the tubers with the highest rate. Thus, further

investigation on the response of potato crops to combined effect of C:N ratio and fertilization rate is

needed.


7

Abbreviations

C: Control

CN; C:N ratio : Carbon to Nitrogen ratio

DAP: Days After Planting

DM: Dry Matter yield

ED: Emergence Date

K: potassium

N: Nitrogen

P: Phosphorus

R: fertilization Rate

WAB: Weeks After Burying

WAP: Weeks After Planting


8

I- Introduction

1) Background and Rationale

In organic plant production, soil fertility is maintained or improved thanks to legumes, organic

amendments and deep-rooting crops. During the growing season, additional fertilizer is often

provided to meet the crops needs and improve the yield and quality. These nutrients can be provided

by animal-based manure and green manure (Sorensen and Thorup-Kristensen, 2011). Previous

studies comparing application of animal manure and mulched green manure on crop fields showed

that animal manure increased the yield, while the application of mulch did not have any significant

effect on yield (Olesen et al., 2009). Therefore, current organic arable farmers are facing problems

with regard to the efficient use of green manure derived-nitrogen.

Organic arable farms in the Netherlands are specialized in high yielding cropping systems such as

potato, sugar beet and onion production (Koopmans and Bokhorst in Canali et al., 2004). These farms

are stockless, meaning there is no animal on-farm which could provide animal-based manure. In the

Netherlands, however, more and more organic farmers are using cattle slurry and solid dairy manure

as their major source of nutrients (Koopmans and Bokhorst in Canali et al., 2004). This comes in

opposition with regulations about fertilizer inputs becoming increasingly strict in organic sector.

Restrictions are made on the amounts of nitrogen and phosphate inputs, purchase of animal-based

manure and use of compost. Moreover, the availability of animal manure from organic process is

limited already. A gradual legislation shift towards a zero use of conventional manure in organic

agriculture is therefore a strong stimulation to find alternatives to animal manure as fertilizer (Burgt

and Bus, 2013).

Mobile green manures, also known as cut-and-carry fertilizers could be a serious new strategy to

supply nutrients on organic arable farms. It is to be distinguished with traditional green manures that

are being harvested and incorporated on the same field to improve soil fertility. Cut-and-carry

fertilizers, on the other hand, are being harvested, transported as a whole, sometimes stored, and

applied and/or incorporated to other fields for fertilizing purposes (Nygaard and Thorup-Kristensen,

2011). Therefore, the nutrients accumulated by the plant (legumes and non-legumes) can be used as

a source of nutrients for the crops needs. As opposed to animal manure, there is no restriction

concerning the use of on-farm produced cut-and-carry fertilizers, which can be an advantage when

the nutrients content is low. Use of such amendment would come in agreement with organic

principles (i.e. closing the nutrients cycles and making the most of on-farm nitrogen dynamics). It

would be even more relevant for stockless organic farmers already providing fodder to breeding

farms, as the economic revenues they get out of it is limited anyway, while the costs of harvesting

and transport can be relatively high (Sørensen et al., 2013; Burgt et al., 2013). Also, with increasing

prices of organic animal-based manure, it will become financially interesting to make use of cut-and-

carry fertilizers.

Previous studies have compared the performances of cut-and-carry fertilizers (fresh and ensiled,

alfalfa and grass clover) with animal manures (cattle slurry, poultry manure, solid cattle manure). It

has shown that cut-and-carry fertilizers nutrient-use efficiency was similar or better, while having a

phosphorus- and potassium-content matching closer with the crops demand. Thus, the

environmental risks such as leaching and volatilization can be minimized. On top of this, crop yields

were similar or better when using cut-and-carry fertilizers (Burgt et al., 2010; Drakopoulos et al.,

2015; Terra, 2014).

Cut-and-carry fertilizers can be applied either fresh or stored by being dried, composted or ensiled

before being applied. Supplemental fertilizers should meet some requirements: it should keep well


9

and be easy to handle during transport, application and incorporation, while being easily available for

the crops (Nygaard and Thorup-Kristensen, 2011). Although it has been shown there were no

significant differences in crop yield between fresh-cut and ensiled mobile green manure (Burgt et al.,

2013), applying big amounts of fresh cut-and-carry fertilizer can cause fungi problems and damage

the crops. Also, storing plant material through wintertime enables farmers to keep the catch crops

derived-nitrogen over a period of time and to use it whenever it is needed during spring of the next

year (Carter et al., 2014).

Grass-clover silage can have a potentially high fertilizer value, depending on the chemical

composition of the plant material. The fertilizer value of mobile green manure does not depend on

the amount of N applied but on the amount of N available. In fact, the release of nutrients by green

manure is influenced by the concentration of carbon in relation with the other nutrients (C:N ratio),

and by the concentration of resistant constituents such a lignin and cellulose (Nygaard and Thorup-

Kristensen 2011 and references within). This is due to the decomposition of plant residues being

undergone by soil microorganisms. They primarily assimilate nutrients for their own growth and

multiplication, before it is made available for the crops under mineral form during microorganisms

turnover; except if the amount applied exceeds their need. When the carbon content of plant

material is high, this may affect the microbial communities and favor fungi development instead of

bacteria.

More precisely, a study about the effect of the chemical composition of plant residues on the crop

yields showed the production of cauliflower and kale decreased when the C:N ratio of applied green

manure increased (Nygaard and Thorup-Kristensen, 2011). A similar study in 2013, with grass-clover

silage this time, confirmed this first result: there is a significant negative correlation between the N

fertilizer value of mobile green manure and the C:N ratio (Sørensen et al., 2013).

Some studies were conducted in 2013 in Wageningen University and Research (Netherlands) on the

use of grass-clover silage as fertilizer, among others (solid cattle manure and Lucerne pellets), for

potatoes and cauliflower (Aloysius, 2013; Drakopoulos, 2014; Terra, 2014). The results showed that

the use of such fertilizer leaded to N-immobilization instead of a net release of plant-available N after

soil application, which was associated with a high C:N ratio of the plant material. Several

recommendations were therefore made for further research on the topic. Therefore, it was

recommended to use grass-clover silage with a higher N content. It was also suggested to apply

grass-clover silage several weeks before planting to ensure a better synchronization between mineral

N release and crop demand. In addition to this, Terra (2014) could not find any significant effect of

grass-clover silage compared to animal manure, which he related to a high initial soil N-content on

field.

In 2014, however, a similar study was conducted on the same experimental farm, where the C:N

ratio from the grass-clover silage was below 20. In this case, use of grass-clover silage resulted in

better crop growth performance, together with a higher total yield and a higher total Apparent

Nitrogen Recovery, in comparison with solid cattle manure and Lucerne pellets (Litsos, 2015).

Also, a 6-year field trial which started in 2012 has been established in the province of Groningen,

Netherlands, in which an innovative agricultural system was based on a 100% on-farm nitrogen

supply. The first year in 2012, a nitrogen immobilization was observed as well, due to a high C:N ratio

(low N content: 0,9% N) (Burgt and Bus, 2012). However, in 2013 where the C:N ratio of grass-clover

silage was 16, the potato yield was much more promising (Hospers-Brands et al., 2014). Therefore,

the scope of this study is to explore the influence of grass-clover silage composition on its capabilities

to respond to the potatoes nutrients needs.

Previous experiments showed that potato tuber yields increased with the application rate (Jenkins

and Nelson, 1991; Sincik et al., 2008; Canali et al., 2010; Sorensen et al., 2013; Terra, 2014; Litsos,


10

2015). In a cropping system, selecting the appropriate nitrogen application rate as part of nitrogen

management is among the first considerations, especially in sandy soils which are more prone to

nitrogen losses. Jenkins and Nelson (1991) studied the effect of three nitrogen fertilizer rates (80,

160 and 240 kg N ha-1) on potato tuber yield. Their research showed there was no significant

difference in yield between medium and highest rate, although there was an increase in plant and

tuber nitrogen content between the two rates. This can be linked to luxury consumption of nitrogen

by the plant, meaning the plant uptook some nitrogen in excess to what is needed to make an

efficient gain in yield. The necessary amount of nitrogen to apply in case of this study (160 kg N ha-1)

actually goes in line with the maximum amount of nitrogen allowed by SKAL in the Netherlands,

which is 170 kg N ha-1. Two more recent studies were conducted in WUR centre, with the following

application rates: 57, 113, 170 kg N ha-1. The first one showed there was no significant difference in

tuber yield between the control plots and the lowest rate, meaning potato plants have a great

capability to uptake nitrogen even in non-fertilized plots (Terra, 2014). The second study showed

there was no significant effect of rate on N recovery (Litsos, 2015). The potato plants were able to

uptake equivalent amounts of N regardless the application rate.

Formulating suitable N rate recommendations are therefore complex and depend on a range of

inherent factors. According to Wolkowski et al. (1995), this choice depends on the crop, the soil

texture, the soil organic matter, the soil yield potential and the yield goal. Choosing an appropriate

rate is therefore dependent on the site conditions where the crop is being grown. This also includes

climate and weather conditions, soil aeration and drainage and soil mineralization capacities (USDA-

NRCS, 2012). When using mobile green manure for fertilization purposes, the length of the growing

season and the mineralization level of the plant material should be taken into consideration. If the

growing season has to be shortened because of a disease outbreak, as it can often be the case in

organic agriculture, the green manure fertilizer value might not be used to its full potential,

whenever the N release comes late in the growing period.

In the current study, further research will be conducted in order to determine the most efficient N

application rate based on agronomical, economic and environmental considerations.

2) Diagram of the research design

The diagram below (Fig. 1) illustrates the research framework of this study. The brown rectangular

sets the boundary of the system, which enclosures all of its components. The blue arrows point at

the variables that will be analyzed within the system. The brown arrows indicate the inputs and

outputs of the system. The green arrows indicate the possible influence of the chosen (i.e.

controlled) factors on the system. The red arrows indicate the influence of external factors on the

system.


11

3) Purpose of the study and Research questions

Organic farmers are currently facing challenges in using green fertilizers while diminishing their

dependence on animal manure. Grass-clover silage was seen as a possible alternative. The challenge

now is to reach a more optimal use of grass-clover silage as cut-and-carry fertilizer.

The objective of this study is to evaluate the influence of the C:N ratio and application rate of grass-

clover silage used as cut-and-carry fertilizer on crop growth performance and tuber yield of organic

potatoes. This has never been investigated in details before.

a. Research question

How do grass-clover silage C:N ratio and its application rate affect the potato growth performance

and yield?

b. Hypotheses

(i) C:N ratio will affect the potato growth performance and the tuber yield, with better growth

performance and higher yields when the C:N ratio is below 20.

(ii) C:N ratio below 20 will allow the potato plants to have a faster access to plant-available nitrogen,

thanks to faster net nitrogen mineralization.

Figure 1. Diagram of the research design

Input: Grass-

clover silage

Output: Potato

tubers

Diseases

Abiotic factors: Temperature, water, wind, …

Pests

Weed

C:N ratio of

silage

Application

rate

Potato plant (canopy, rooting system)

Soil mineral content Soil

Biological life

(worms, bacteria, fungi, …)

Canopy

volume

Light

interception

Dry matter yield

Tuber yield Plant height

Tuber N content


12

II- Material and methods

1) Experimental site: Droevendaal farm

The experiments were conducted at the Droevendaal farm, the experimental farm of Wageningen

University and Research Centre in Wageningen, the Netherlands. It is a Skal-certified organic

research facility of 50 hectares. The climate on the experimental site is temperate oceanic with an

annual mean temperature of 11°C and annual cumulative precipitations of 829 mm. The soil type is

classified as silty-sand. An 8-year crop rotation has been established on the farm, including winter

triticale, spring wheat, spring barley and rye, temporary and permanent pastures of grass-clover,

potatoes, field beans, cabbage, and an orchard along with cover crops. The crops in place until the

experiment were catch crops (with no legumes) consisting of rye and mustard mainly. The previous

crop on the experimental field was spring barley and spring wheat. Weather data (i.e. minimum and

maximum averaged weekly temperatures and cumulative weekly rainfall) during the potato

production period were collected from a local weather station (Fig. 2).

Figure 2. Cumulative weekly precipitations and minimum (Tmin) and maximum (Tmax) averaged weekly

temperatures during potato growing cycle (22/04/2015-10/09/2015).

2) Experimental design and management

a. Experimental design

The chosen experimental design is a Randomized Complete Block Design (RCBD) with 4 replicates.

There are two factors in this experiment: the C:N ratio and the nitrogen application rate. The first

factor has 4 levels while the second one has 4 levels (when including control) (Table 1). A non-

fertilized plot was added per block as a control treatment, which was used later for agronomic

response and N uptake efficiency calculations. There are in total 13 treatments that were randomly

disposed per block, which results in 52 plots, as shown in the Figure 3 below. The plot #0 is a

supplementary control plot that was used for some extra-measurements. The experimental unit is a

plot of 15 m x 3 m (45m²), with 4 rows of potatoes.

0,0

5,0

10,0

15,0

20,0

25,0

30,0

35,0

0,0

10,0

20,0

30,0

40,0

50,0

60,0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Av

era

ge

te

mp

era

ture

(°C

)

Cu

mu

lati

ve

we

ek

ly p

reci

pit

ati

on

s

(mm

/we

ek

)

Weeks after planting (WAP)

Precipitations

Tmin

Tmax


13

Table 1. Levels of each factor

Factor C:N ratio Nitrogen fertilization rate (kg N ha-1)

Level 24 0 (Control)

22 57

17 113

16 170

Table 2. Treatments list (legend of Figure 3)

1 Control

Field # C :N

ratio

Application rate

(kg N ha-1)

0 0

1 24 57

2 24 113

3 24 170

4 22 57

5 22 113

6 22 170

7 17 57

8 17 113

9 17 170

10 16 57

11 16 113

12 16 170

13 C1 0

N

Figure 3. Experimental layout


14

b. Agronomic practices

The grass-clover was mowed 4 times in 2014 and dried in the field during one to three days before

being compressed in round bales and wrapped in plastic film to be ensiled throughout winter. The

previous crops were mowed and incorporated several days before grass-clover silage application.

One week before planting, grass-clover silages were chopped and weighed before being broadcasted

by hand on each plot, to be evenly distributed, with the targeted amounts of 57; 113 and 170 kg N

ha-1. Grass-clover silage was incorporated to the soil through ecoplough, to a depth of 15 cm with a

shallow moldboard machine. Ecoploughing allowed mixing the organic amendment with the soil,

loosening the soil, limiting the amount of clods, and helping prepare properly the seeds bed. The

seed potatoes were from the Carolus variety, which is an organic variety that is resistant to

Phytophtora infestans in foliage and tuber (Agrico, 2015). They were planted on the 22nd April 2015,

6 days after fertilizer application, with an inter-row spacing of 0,75 m and an intra-row spacing of

0,29 m (as recommended by the breeder Agrico Research BV) (Agrico, 2015). The ridges were formed

at the same time, and re-ridging occurred twice during the growing season, the 20th may and the 15th

june, also for weed control.

It was not possible to follow the recommendation of the previous studies which consisted in applying

grass-clover silage several weeks before planting (Drakopoulos, 2015; Litsos, 2015), as the soil

temperature by that time of the year was too low on the experimental site to ensure a proper

decomposition of plant material by the soil biota.

3) Preliminary experiments

In order to select the grass-clover silages with a suitable C:N ratio for this experiment, grass-clover

silages produced on the experimental farm were analyzed on 11th march 2015. Samples were oven-

dried at 70°C during 24h and grinded with a 1-mm sieve to determine the dry matter content, the

organic matter (OM) content, the ash content, and total Carbon (total C) and Nitrogen (total N)

contents (all in %), in order to get the C:N ratio of each of the silages. Ash content and organic

matter were measured using the loss-on-ignition (LOI) methodology described by Konare et al.

(2010) through combustion of the samples in a furnace at 500-550 °C for 3 hours. Total C content

was determined using the Dumas Method with a CHN1110 Element Analyser (CE instruments, Milan,

Italy). Total N content was determined following the methods described in Houba et al. (1999) (more

details in paragraph below). The results can be found in the Table 3 below.

Table 3.Chemical composition of grass-clover silages (11th march analysis)

The initial soil nutrient composition was analyzed according to the following. 30 soil sub-samples

were taken per block thanks to a soil auger, on a depth of 0-30 cm according to a zig-zag pattern,

before any fertilizer application. Considering the soil is rather homogeneous within each block, the

soil sub-samples were then mixed together to form one single composite soil sample per block,

Samples were oven-dried at 70°C during 24h before being analyzed in order to determine the DM

content, the OM content, the ash content (all in %), the total N, P, K and the plant-available macro-

nutrients, i.e. total mineral N (Nmin), P2O5 and K2O (all in g kg-1) and the pH. OM content and ash

content were analyzed in the same way as previously mentioned for silage samples. Total N, total

Silage # Harvest time DM (%) OM (%) Ash (%) Total C (%) Total N (%) C/OM C/N

1 May 76 91 9 42 1,6 0,46 27

2 June-July 60 91 9 43 2,2 0,48 20

3 Aug-Sept 45 90 10 44 2,8 0,49 16

4 Oct-Nov 16 80 20 41 3,1 0,51 13


15

Nmin, total P and P2O5-P were determined in the same way as total N in the silage samples: following

the methods described in Houba et al. (1999),samples were dried at 40 ο C, extracted in 0,01M

calcium chloride and analyzed using a segmented-flow system (Auto-analyzer II, Technicon). In the

same extracts, total K and K2O-K were measured with a Varian AA240FS fast sequential atomic

absorption spectrometer (Terneuzen, the Netherlands). The pH was measured using a pH/mV meter

(Inolab pH/Cond Level 1, WTW, Weilheim, Germany). The results can be found in the table below

(Table 4).

Table 4. Soil characteristics on the experimental field before any fertilizer application

Block # DM OM ash Total N Total P Total K Total Nmin PO43--P K2O-K pH-H2O

(%) (%) (%) (mg g-1) (mg g-1) (mg g-1) (mg g-1) (mg g-1) (mg g-1)

1 87 2,9 97,1 0,8 0,6 - 0,00 0,00 0,05 6,3

2 86 3,8 96,2 0,9 0,6 - 0,00 0,00 0,05 6,1

3 86 3,8 96,2 0,9 0,6 - 0,00 0,00 0,04 6,1

4 86 3,8 96,2 0,8 0,6 - 0,00 0,00 0,04 6,1

Estimations were done to determine the amount of fertilizer to apply in accordance with the silage

and soil nutrients contents and the target nitrogen application rates (kg N ha-1). In order to have an

idea of the fertilizer value of the grass-clover silages, 10 sub-samples were taken from each silage

the day of their application, the 16th april. Samples were oven-dried at 70°C during 24h and grinded

with a 1-mm sieve in order to determine the DM content, the ash content, the OM content (all in %),

the pH, and the nutrients content of the grass-clover silage, i.e. total N, P, K, NO3- -N, NH4

+-N, P2O5

and K2O (all in g kg -1) at the time it was applied to the soil. The methodologies were the same as

previously mentioned for soil samples. The C:N ratio was then recalculated, and the results from

both analysis were averaged for the rest of the experiment (Table 5) (see details in Appendix 1).

Table 5. Chemical composition of grass-clover silages (averaged over the two laboratory analyses)

Silage # Harvest

time

DM

(%)

OM

(%)

Ash

(%)

C

(g/kg)

N

(g/kg)

C:N P

(g/kg)

K

(g/kg)

Total

Nmin

(g/kg)

NH4-N

(g/kg)

NO3-N

(g/kg)

PO4-P

(g/kg)

K20-K

(g/kg) pH-H2O

1 May 73 91 9 436 17,8 24 3,6 30,9 0,66 0,65 0,00 3,2 31 5,7

2 June-July 64 91 9 442 19,7 22 3,4 30,5 0,68 0,68 0,00 2,9 36 5,4

3 Aug-Sept 47 89 11 460 27,0 17 4,4 35,8 0,80 0,79 0,01 4,1 37 4,9

4 Oct-Nov 19 78 22 412 26,3 16 4,4 24,5 0,65 0,65 0,00 3,7 34 4,5 1 all expressed in dry weight

From Table 5, one can observe decreasing DM content, increasing ash content, increasing C content

– except for silage #4 – and increasing N-P-K contents over the 4 cuts. In fact, carbon content of

grass-clover pastures depends on the combined effect of temperatures and percentage of

reproductive materials, namely stems and flowers. The summer during the growth of pastures was

characterized by high temperatures, and the 3 first cuts between May and September had a lot of

stems. In late autumn however, grass had mainly leaves and not much stem, so that lignin content

and subsequently C content was lower. Also, the high ash content from the last cut was probably due

to soil contamination.


16

4) Field measurements and laboratory analysis

As there was no distinct separation between the plots, a net plot has been delimitated within each

plot, in which all the measurements were done, to avoid border effects. For that matter, the two

external rows and the 3 first and 3 last plants from the two internal rows were not used for

measurements. The 6 next potato plants from the two internal rows, situated after the 3 first and

before the 3 last plants were left for destructive measurements. Consequently, the overall net plot

consisted of 2 rows of 34 plants each.

a. 50% plant emergence date

As soon as the first plants were visible on the field, the number of plants that have emerged was

counted per net plot within 2 days interval, until more than half of the expected number has

emerged within a net plot. Then, the 50% emergence date was calculated, thanks to the following

formula:

Formula of 50% plant Emergence Date (ED):

ED= �1 + ��2 − �1� ∗ �� (1)

Where:

T: days after planting

(T1: where <50% have emerged)

(T2: when ≥ 50% have emerged)

Target: 50% of the expected number of potato plants based on planting density

N: number of plants emerged

(N1: at T1)

(N2: at T2)

b. Plant growth performance

From the previous studies, it was shown there was a significant correlation between plant height and

canopy volume with final yield (Drakopoulos, 2015; Litsos, 2015). Therefore those measurements are

good predictors of the final yield. On the contrary, it was shown several times there was no

significant correlation between the leaf chlorophyll index measured with a SPAD meter and the final

yield (Drakopoulos, 2015; Litsos, 2015). The leaf area index, however, had a significant correlation

with final yield (Litsos, 2015). Other research studies showed this index was strongly correlated with

light interception. The potato tuber yield is indeed closely related to the ability of the plant to

intercept solar radiation (Boyd et al., 2002). Therefore, light interception was also measured as a

predictor of the final yield.

Plant height, canopy volume and light interception were measured the same week, every two weeks,

starting from when most of the plants had emerged in the net plot, and until no differences in height

and volume could be seen visually between the plots. Therefore, measurements were done at 7, 9,

and 11 WAP (weeks after planting).

For plant height and canopy volume, measurements were taken on a plant every 3 steps

(corresponding to 2 meters) and alternatively on the left and right ridge, so that 5 plants were

measured per net plot.

Plant height

The plant height is the distance in cm between the soil level and the highest point of the above-

ground crop.


17

Canopy volume

The canopy volume was calculated based on the formula of an elliptical cone volume, as seen below

(Drakopoulos, 2015):

Formula of canopy volume (CV): CV = �∗��∗�

� (2)

Where:

D: plant diameter, which is the average of plant length and width in cm.

H: plant height in cm

Light interception

The light interception is the difference in total radiation measured from above and below the

canopy. It was calculated thanks to the following formula:

Interception (%) = ��

�� * 100 (3)

Where :

Total : total incident radiation measured above the canopy

PAR: Photosynthetically Active Radiation measured below the canopy

This was done thanks to a SunScan Canopy Analysis System or tube solarimeter, which measured the

light intercepted below the canopy –thanks to a portable probe- and above the canopy – thanks to a

reference sensor placed close to the field. For each plot, 10 measurements were done, using 5

plants. Starting from the North side of the net plot, the 5 first plants from one row were taken, in

order to avoid walking too much in between the rows and creating compaction. For each of the 5

plants, the 10 measurements consisted of one reading under the canopy, close to the stem, and one

reading in between 2 plants. Each reading was done with the sunscan probe being placed in a

horizontal plane, pointing the West-East direction and placed at a right angle to the row. The end-

part of the probe was placed in the middle of the inter-row so that the middle-part of the probe was

situated in the middle of the row. The length of the probe was readjusted to the distance between 2

rows (0,75m). Readings were done between 10:00 and 15:00 when possible, during which the light

conditions had to be the same. Due to slight but consistent differences in readings between the

sunscan probe and reference sensor, the two sensors were calibrated in between each block by

taking 10 reference measurements next to one another. A correction factor was subsequently

calculated and the PAR readings were corrected by this factor.

c. Nitrogen release from grass-clover silage

The nitrogen dynamics in the decomposing silage were quantified using litterbags (20 x 15 cm). Their

mesh size was 1mm to exclude access to earthworms, as their distribution on the top 30 cm soil

depth is heterogeneous, and that would avoid having too much soil contamination in the litterbags

through their excretion. For each block, 24 litterbags (4 bags containing each a different C:N ratio,

repeated 6 times for 6 excavations) were filled with 30g of silage and randomly buried horizontally

within the rows of the 14th extra-plot for each block (F0, see Figure 3) on the 16th may. The litterbags

were placed at a 15-cm depth, between the potato plants, with 1,5m spacing, to ensure they were

placed at an equal distance within the rows.

100g of silage from each C:N ratio were taken the day the litterbags were filled to determine the

silages’ initial composition. Samples were oven-dried at 70°C during 24h and grinded with a 1-mm

sieve before being analyzed to determine the dry matter content (%DM), the total nitrogen and

carbon content (all in g kg-1), according to the same methodology as previously mentioned.

At 1, 2, 4, 8, 12 WAB (weeks after burying) and on the day the potato canopy was burnt, 15 WAB,

one silage litterbag from each treatment were excavated randomly. The litterbags were emptied and

the remaining silage was oven-dried at 70°C during 24h and grinded with a 1-mm sieve, to determine


18

the dry matter content (%DM) and the total Nitrogen content (g kg-1), using the same methodologies

as previously mentioned.

Nitrogen release from grass-clover silage was evaluated overtime through the measure of dry weight

and total N disappearances from the plant material, according to the following formula (Cobo et al,

2002):

N released (%) = � �!

* 100 (4)

Where:

Nt: amount of total N released at the excavation time t (mg/g)

N0: initial N content (mg/g) in grass-clover silage

The dry weight and nitrogen content were corrected for soil contamination thanks to ash contents

and using the following formula, according to Cusick et al. (2006):

SC= �"#$��"%&

'( (5)

Where:

SC: dry weight of soil contamination (g),

ACLB: ash content of the total remaining material in the litterbag (mg),

ACSI: initial ash content of the grass clover silage (mg),

ACSO: ash content of soil (mg g−1)

The main assumption here is that the ash content in the silage remains the same during

decomposition.

d. Tuber yield and yield components

In case of this study, the potato tubers were grown to be sold to a seed potato company. The

potatoes were considered to be ready to harvest when half of the tubers from one plant had a

caliber between 40 and 60 mm. At 20 WAP, on 10th September, the harvest was done manually on a

10m² area centered within the net plot. The tuber fresh weight of each plot was measured in order

to determine the total fresh weight (ton ha-1). Afterwards, two tuber subsamples of 0,5 kg and 0,1 kg

were collected in order to determine respectively tuber DM (%) and total N content (%).

Thereafter, some indicators of performance were calculated.

The agronomic efficiency (AE, in kgyield01��Napplied) was calculated thanks to this formula:

56 = 89:;<=>:?=@:A=−89:;<BCA=>C;D?EE;9:< (6)

Where:

Yield treatment: total yield (kg ha-1) for one treatment

Yield control: total yield (kg ha-1) for the control (averaged over the 4 blocks)

N applied: amount of nitrogen applied (kg N ha-1)

This is an indicator of the increase in yield for every unit of N added.

The nitrogen accumulated (N accumulation, in kg N ha-1) and the apparent N recovery (ANR) were

also determined in the tubers, thanks to the following formulas:

N accumulation= DM yield * tuber total N content (7)


19

ANR in the tubers = NaccumulationLMN OPOQMRSPT –Naccumulationcontrol

D?EE;9:< *100 (8)

Where:

DM yield (kg ha-1) = Fresh tuber yield (kg ha-1) * tuber dry weight (%)

Tuber total N content in %

ANR is an indicator of the efficiency by which the fertilizer N applied is recovered by the crop.

5) Statistical analysis

The data (in Appendix 2) was analyzed using the software R (3.0.2). An ANalysis Of Variance (ANOVA)

was conducted with a linear model, to test the effects of C:N ratio and fertilization rate on the

response variables, with a significance level of 0,05. Interaction between C:N ratio and fertilization

rate was included in the model, as well as the factor block, so that variation explained by the blocks

were taken into account in the calculations of the sums of square in the ANOVA. ANOVA assumptions

on the residual data were assessed using Shapiro-Wilk test and Levene’s test to check their normal

distribution and homogeneity of variance. When effect was significant, post hoc test Tukey HSD was

done to do multiple pairwise comparisons. Also, a simple linear regression analysis was conducted in

order to test the linear relationship between fertilization rate and each of the response variables (as

it was assumed 57 and 113 correspond to 1/3 and 2/3 or 170 respectively), with a significance level

of 0,05.

For light interception, measurements taken over time at 7, 9 and 11 WAP were repeated on the same

individual plants, causing temporal pseudoreplication, so that data from one measuring time to

another don’t have independent errors. This is a violation of one of the main assumptions for

statistical analysis. Therefore, in addition to the previously mentioned ANOVA, a second ANOVA was

conducted with a mixed-effects model, where time of measurements (in weeks after planting) was

taken as a random effect repeated on each experimental unit. This allowed to check the main effect

of C:N ratio and fertilization rate on light interception over the 5 weeks of measurements.

Violation of normality was not taken into account as literature often states that an ANOVA is not very

sensitive to moderate deviations from normality (Glass et al., 1972; Harwell et al., 1992; Lix et al.,

1996). Violation of homoscedasticity was also not taken into account, as transformation of data (log,

ln, square root and arcsin) did not overcome the problem. However, being aware that using a

parametric test when assumptions are violated might increase chance of false positive results,

violation of ANOVA assumptions (if any) will be mentioned in the table of results.

Including the control treatment as a level of fertilization rate rendered the design unbalanced.

Therefore, main effect of fertilization rate was assessed separately, with a two-way ANOVA

(including factor block), where means by fertilization rate were averaged across C:N ratios.


20

III- Results and discussion

1) Conditions during growing season

During the potatoes growing period, the temperature averaged 15,9°C and the cumulative

precipitations were 216 mm (Fig. 2). In June especially, temperatures were abnormally high for the

Dutch season. No Phytophthora outbreak was observed and some Colorado beetles did minor

damages only. Weeds also benefited from the good climatic conditions but it was not detrimental to

the potato plants. Therefore, on the overall the conditions were optimal for this growing season.

2) Plant growth: emergence date, plant height, canopy volume, light interception

a. Plant emergence

50% of the plants amended with CN22 emerged one day later (40 DAP) than 50% of the plants

amended with the three other C:N ratios (39 DAP). With fertilization rate, 50% of the plants from

plots amended with the highest rate (i.e. 170 kg N.ha-1) emerged one day earlier (39 DAP) than plants

amended with 57 and 113 kg N.ha-1. However, in both cases results were not significantly different

(Table 6).

This is a rather late emergence date. However, re-ridging occurred during the emergence stage, at a

time when 15% of the plants per plot had already emerged on average. This caused complete cover

by the soil of the emerged plants, and the newly formed ridges were taller than the ones formed at

planting. Therefore, this might have delayed the time of emergence. On the other hand, making

ridges taller allow the tubers to expand (Navarre and Pavek, 2014).

Also, variations in plant emergence were observed within each plot, with up to 4 weeks difference

between the first and the last emerged plants. This can have several reasons. This might be due to

the variety itself, given the fact that other factors that might have influenced plant emergence were

supposed to be similar among the seed tubers: same physiological age, similar size and similar soil

conditions at and after planting.

So, C:N ratio and fertilization rate did not affect emergence time. Although published literature on

the effect of fertilization application on emergence is scanty, these results go in line with Trehan et al

(1998 in Govindakrishnan and Haverkort, 2006) who found there was no effect of N on emergence.

This can be explained by the source of energy the sprout is using to emerge from the soil. During the

establishment phase, the sprout uses mainly the energy coming from the mother seed tuber

(Govindakrishnan and Haverkort, 2006).

b. Plant height

Potato plants from non-amended plots had consistently the smallest height over the 3 measuring

times, therefore showing there was an effect of grass-clover silage as fertilizer on the plant height

(Table 6). At 7 WAP, plants had similar heights across C:N ratios and fertilization rate, so there was no

significant effect of the factors on the plant height. This is consistent with the visual observations

that were made, where big variations in plant heights could be seen within each plot, due to late

emergence. In fact, there was a large variation both in emergence and height between plants within

one plot in the early stages of plant growth. At 9 WAP, potato plants fertilized with CN16 and CN17

were significantly taller (60,9 and 60,8 cm respectively) than with CN22 and CN24, who had similar

heights (56,3 and 54,7 cm respectively) (Table 6). Actually, there was somewhat a tendency to linear

decrease in plant height as C:N ratio increased, as shown on Figure 4. Fertilization rate did not have

any effect at 9 WAP (Table 6). However at 11 WAP, whereas C:N ratio had no effect on plant height

anymore, there was a positive linear relationship with fertilization rate (Table 6 and Figure 5). Potato

plants from non-amended plots and lowest rate (R57) were the shortest while potato plants fertilized

with the highest rate (R170) were the tallest, and plant fertilized with R113 had intermediate values.


21

This is also consistent with visual observations. Differences among each plot disappeared while some

differences between treatments could be seen from a visual inspection at 9 WAP already.

10

20

30

40

50

60

70

80

0 50 100 150 200

Pla

nt

he

igh

t (c

m)

Fertilization rate (kg N ha-1 )

10

20

30

40

50

60

70

80

0 50 100 150 200

Pla

nt

he

igh

t (c

m)

Fertilization rate (kg N ha-1)

10

20

30

40

50

60

70

80

0 50 100 150 200

Pla

nt

he

igh

t (c

m)


(a) (b) (c)

Figure 5. Plant height at 7 (a), 9 (b) and 11 (c) weeks after planting as affected by N fertilization rate.

Error bars stand for standard errors. The dashed line stands for linear trend.

15

25

35

45

55

65

75

15 20 25

Pla

nt

he

igh

t (c

m)

C:N ratio

15

25

35

45

55

65

75

15 20 25

Pla

nt

he

igth

(cm

)

C:N ratio

15

25

35

45

55

65

75

15 20 25

Pla

nt

he

igh

t (c

m)

C:N ratio

(a) (b) (c)

Figure 4. Plant height at 7 (a), 9 (b) and 11 (c) weeks after planting as affected by C:N ratio.



22

c. Canopy volume

Regarding canopy volume, both C:N ratio and fertilization rate did not have a clear effect at 7 WAP

(Table 6). This is consistent with the visual observations: variations in canopy volume within each plot

were even more noticeable than in plant height. At 9 WAP, plants fertilized with CN16 resulted in

significantly bigger volumes than plants fertilized with CN24, while CN17 and CN22 had intermediate

values. There was actually a tendency to linear decrease in canopy volume as the C:N ratio was

increasing, as shown on Figure 6.

Fertilization rate did not have a clear effect on canopy volume at 9 WAP (Table 6). At 11WAP

however, potato plants fertilized with the highest rate (R170) resulted in having significantly bigger

volumes than plants fertilized at lower rates (R0, R57 and R113). Also, there was a significant positive

linear correlation between fertilization rate and canopy volume at 11 WAP (Table 6 and Figure 7).

0

20

40

60

80

100

120

140

15 20 25

Ca

no

py

vo

lum

e (

x1

03

cm

3)

C:N ratio

0

20

40

60

80

100

120

140

15 20 25

Ca

no

py

vo

lum

e (

x1

03

cm3)

C:N ratio

0

20

40

60

80

100

120

140

15 20 25C

an

op

y v

olu

me

(x

10

3cm

3)

C:N ratio

(a) (b) (c)

0

20

40

60

80

100

120

140

0 50 100 150 200

Ca

no

py

vo

lum

e (

x1

03

cm3)


0

20

40

60

80

100

120

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0 100 200

Ca

no

py

vo

lum

e (

x1

03

cm3)


0

20

40

60

80

100

120

140

0 50 100 150 200

Ca

no

py

vo

lum

e (

x1

03

cm3)


(a) (b) (c)

Figure 6. Canopy volume at 7 (a), 9 (b) and 11 (c) weeks after planting as affected by C:N ratio.


Figure 7. Canopy volume at 7 (a), 9 (b) and 11 (c) weeks after planting as affected by N fertilization rate.



23

Thus, plant height and canopy volume showed similar results. C:N ratio has an effect on early stage

of canopy growth (although it was not significant at 7 WAP), and there is a significant difference

between higher C:N ratios and lower C:N ratios. I fact, plant growth is proportional to the amount of

N in the plant, so that N deficiencies limit canopy growth. Therefore this significant difference could

be linked to a different trend in net N mineralization during first stages of decomposition by soil

microorganisms. Whitmore (2007 in Canali et al., 2012) said it was generally accepted that easily

decomposable plant material with a C:N ratio below 20 would release N on decomposition while

material with C:N ratio above 20 would immobilize N temporally.

Also, fertilization rate has a significant effect at a later stage of canopy growth, when full canopy

cover was almost reached. This suggests N immobilization was overcome 2 weeks later. Increasing

amounts of nitrogen applied increases linearly the growth performances of the plants, with a 25%

increase in canopy volume between highest rate and other rates.

One can argue about the representativeness of chosen plants within a plot, so that 5 other plants

used for the measurements might have led to different results. However, those results come in line

with other measurements done during this experimental study (Palomba, 2016). It was shown that

CN24 resulted in a significantly lower amount of N accumulated in the aboveground biomass and

tubers at 10WAP, which was not the case anymore at 14 WAP. This confirms the hypothesis of N

immobilization.

As a remark, this time, due to a much longer growing season in comparison with Drakopoulos (2014)

and Litsos (2015), plant height and canopy volume could no longer be taken as predictors of final

potato yield.

Table 6. Mean values and analysis of variance for the effect of C:N ratio and fertilization rate (kg N.ha-1) on

emergence date, plant height and canopy volume of potatoes at 7, 9 and 11 weeks after planting (WAP).

1 Days after planting

2 *, ** and *** refer to p-values < 0.05, < 0.01 and < 0.001, respectively; ns = not significant.

3 Values within columns followed by different letters are significantly different. p-value < 0.05.

4 Crosses refer to cases when assumptions of normality and homogeneity of residual variances are violated.

Emergence

date

Plant height Canopy volume

7WAP 9WAP 11WAP 7WAP 9WAP 11WAP

DAP1 ------------- cm --------------- ----------.103 cm3-------------

C :N ratio (CN)

Significance ns2 ns ** ns ns ** ns

16 39 22 61 b3 68 9 62 c 112

17 39 22 61 b 70 8 57 bc 101

22 40 19 56 a 68 6 46 ab 98

24 39 21 55 a 66 7 40 a 97

Fertilization rate (R)

Significance ns ns ns ** ns ns **

0 (Control) 39 19 54 63 a 7 39 79 a

57 40 21 57 67 a 7 50 93 a

113 40 21 59 67 ab 7 52 95 a

170 39 22 59 71 b 8 52 118 b

Linear ns ns ns ** ns ns ***

CN*R ns ns ns ns ns ns ns

Normality X4

Error var. x x


24

d. Light interception

C:N ratio and fertilization rate did not have any clear effect on light interception at 7 WAP and 9 WAP

(Table 7). At 11 WAP, C:N ratio had an influence on light interception with highest ratios accounting

for the highest light intercepted (Table 7 and Figure 8). This is not consistent with results obtained

from the other plant growth measurements. This non-consistent evolution over time might not be

due to the controlled factors, but to external non-controlled factors, or to measuring errors. Actually

at 10 WAP, many potato plots all over the field were falling over because the root system and tubers

were not deep enough in the ridge. Therefore one week later, potato canopies grew vertically from

the middle of the ridge, and the plants took an sort of inversed bell shape. This rendered the

measuring complicated as the plants were overlapping each over from one row to another, while not

many leaves were left on top of the ridge. Therefore, the methodology used was not optimal for

measuring light interception in this specific context. Also, one important point to consider is the

foliage structure of the potato plant. The light intercepted by the leaves is heterogeneous along the

shoot, as leaves at the top tend to intercept more light than the ones at the bottom. Therefore,

placing the solarimeter at the bottom of the canopy might result in wrong estimations of light

interception.

Still, on the overall there was an effect of fertilization rate over the whole 5-week time interval, with

the highest rate R170 resulting in significantly higher light intercepted by the potato canopy, as

compared with the two other fertilization rates (R57 and R113). There was an increasing linear

relationship between light interception and fertilization rate over the time interval considered (Table

7).

Table 7. Mean values and analysis of variance for the effect of C:N ratio and fertilization rate (kg N.ha

-1) on

light interception of potato canopy at 7, 9 and 11 weeks after planting (WAP); and over the 5 week period.

Light interception

7 WAP 9 WAP 11 WAP Overall

--------------------------------------------------- % --------------------------------------------

C:N ratio (CN)

Significance ns1 ns * ns

16 19 69 80 a 56

17 22 71 85 ab 59

22 18 66 85 b 56

24 20 68 85 b 58


Significance ns ns ns **

0 (Control) 19 63 a2 79 54

57 18 65 a 83 56 a

113 19 67 a 86 57 a

170 23 73 b 83 60 b

Linear trend ns ns ns ***

CN*FR ns ns ns ns 1 *, ** and *** refer to p-values < 0.05, < 0.01 and < 0.001, respectively; ns = not significant.



25

Figure 8. Light interception over time as affected by treatments combining C:N ratio and N fertilization rate.

CN indicates level of C:N ratio while R indicates level of fertilization rate (kg N ha-1

). Dashed lines stand for

polynomial trend.

3) Yield components: fresh yield, dry matter yield, agronomic efficiency and tuber N

accumulation

a. Fresh yield

First of all, non-amended plots resulted in the lowest fresh tuber yield, which means that N

application through grass-clover silage had a significant effect on final yield (Table 8). In fact, using

grass-clover silage as cut-and-carry fertilizer resulted in a fresh yield of 41 ton.ha-1 averaged across all

the treatments, which represents a 35% increase as compared to non-amended plots. This is a rather

correct fresh tuber yield for organic potato system, as the average potato yields in Dutch organic

farming systems can vary from 12 to 35 ton ha-1 according to Lammerts van Bueren (2010). Then, C:N

ratio had a significant effect on fresh yield, with lowest C:N ratio having a significantly higher yield

than the two highest C:N ratios (i.e. CN22 and CN24), which had similar yields (Fig. 9a).Thus, use of

grass-clover silage with a low C:N ratio (i.e. 16) resulted in a 9-11% increase in tuber yield as

compared with silages having a C:N ratio above 20.

There was a highly positive significant linear correlation between fresh potato yield and fertilization

rate (Fig. 9b). The highest rate resulted in significantly higher yield than medium and lower rates (i.e.

R113 and R57), while control treatment resulted in the lowest yield (Table 8). This means there is

room for higher yield with higher rate applied. This comes in agreement with some studies showing

that there was a significant increase in potato yield between a rate of 80 and 160 kg N ha-1 (Jenkins

and Nelson, 1991). However, this study also showed there was no significant increase in yield

between a rate of 160 and 280 kg N ha-1. Our results are somewhat different to what has been found

in previous experiments in the same study location with the same N application rates (Litsos, 2015).

Differences might lie in the conditions of the growing season which were optimal and resulted in a

longer crop cycle whereas Phytophthora outbreak during the abovementioned study resulted in the

shortening of the growing cycle. This echoes to what was stated earlier in the introduction, that it is

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

90,00

100,00

7 9 11

Lig

ht

inte

rce

pti

on

(%

)

Time (weeks after planting)

CN16-R57

CN16-5113

CN16-R170

CN17-R57

CN17-R113

CN17-R170

CN22-R57

CN22-R113

CN22-R170

CN24-R57

CN24-R113

CN24-R170

C-R0


26

rather difficult to predict prior to planting the effect of rate on final yield. It depends on the weather

conditions during growing cycle.

The highest yield of 50t.ha-1 was obtained from the treatment with highest rate and lowest C:N ratio

(Fig. 10). It is the best combination regarding final yield. Therefore, further research should be done

on the combined effect of increasing rates and decreasing C:N ratios.

Figure 10. Fresh tuber yield as affected by combined treatments of C:N ratio and fertilization rate (kg N ha

-1).

C:N ratio levels: 16; 17; 22; 24. Fertilization rate levels: 0; 57; 113; 170 kg N ha-1

. C-0 is the control treatment.

Error bars stand for standard errors.

0,00

5,00

10,00

15,00

20,00

25,00

30,00

35,00

40,00

45,00

50,00

55,00

C 16 17 22 24 16 17 22 24 16 17 22 24

0 57 113 170

Fre

sh t

ub

er

yie

ld (

ton

.ha

-1)

Treatment

(a) (b)

0,0

10,0

20,0

30,0

40,0

50,0

15 17 19 21 23 25

Fre

sh t

ub

er

yie

ld (

ton

.ha

-1)

C:N ratio

0,0

10,0

20,0

30,0

40,0

50,0

0 50 100 150 200

Fre

sh t

ub

er

yie

ld (

ton

.ha

-1)

Fertilization rate (kg N.ha-1)

Figure 9. Fresh tuber yield as affected by C:N ratio (a) and N fertilization rate (b).



27

b. Dry matter yield

C:N ratio did not have any significant effect on tuber DM yield. However in terms of numerical values,

this comes in line with the fresh potato yield, as it shows a negative trend (Table 8). Fertilization rate

had a significant effect on tuber DM yield, with highest rate (i.e. R170) resulting in significantly higher

DM yield than medium and lowest rates (i.e. R113 and R57 respectively). The relationship between

DM yield and fertilization rate showed a highly significant linear trend (Table 8).

c. Agronomic efficiency

The agronomic nitrogen efficiency was affected by C:N ratio, with CN16 resulting in significantly

higher AE than CN24, while CN17 and CN22 had intermediate values (Table 8 and Fig. 11) . This

means the highest yield increase per unit of N applied was reached with grass-clover silage having a

C:N ratio of 16. Fertilization rate, in turn, did not influence the agronomic efficiency.

Figure 11. Agronomic nitrogen efficiency as affected by C:N ratio.

Error bars stand for standard error.

d. Tuber N accumulation

C:N ratio did not have a significant effect on tuber N accumulation (Table 8). There was only a

tendency to decreasing values with increasing C:N ratio. Given the fact that 10 days before harvest,

all the plants in the field were yellow and dried out, it might be assumed that nearly all of the canopy

N was translocated to the tubers before harvest. Those results come in line with values obtained

from plant height and canopy volume, where C:N ratio influence was significant at 9 WAP but not

anymore at 11 WAP (Fig. 4 and 6), meaning differences became smaller during the last weeks of

canopy growth. It comes also in line with the results found by Palomba (2015) that were previously

mentioned. In fact, the 9th, 10th and 11th WAP were characterized with abnormally high

temperatures for Dutch standards and low rainfall (Fig. 2) which caused an increase in soil

temperature from 15 to 19°C in a few days only (data not shown). Thus, higher temperatures at this

period might have enhanced potato plant growth and development. MacDonald et al. (1995, in

Walley) studied the effect of temperature on microbial activity and N mineralization and found that

accumulation of released N increased with increasing temperatures. Finally, this is consistent with

the values obtained for fresh tuber yield, where the difference in yield between lowest C:N ratio and

higher C:N ratios (i.e. CN22 and CN24) is 10% only (Table 8).

Fertilization rate had a significant influence on tuber N accumulation, with highest rate being

responsible for the highest amount of N accumulated in the tubers. In fact, there was a significant

positive linear relationship between tuber N accumulation and fertilization rate (Table 8 and Figure

0

20

40

60

80

100

120

140

160

15 17 19 21 23 25

Ag

ron

mic

nit

rog

en

eff

icie

ncy

(kg

.kg

N-1

ap

pli

ed

)

C:N ratio


28

12). This is not in line with the results from Litsos (2015). However, again, the longer growing period

for this study might have allowed the potato plants to accumulate more N that was further made

available overtime.

Figure 12. Tuber nitrogen accumulation as affected by C:N ratio.


Table 8. Mean values and analysis of variance for the effect of C:N ratio and fertilization rate (kg N ha

-1) on

fresh tuber yield, agronomic efficiency (AE), dry matter yield (DM yield), tuber nitrogen accumulation (N

accumulation) and tuber apparent nitrogen recovery (ANR) of potatoes.

Fresh Yield AE DM yield N accumulation ANR

ton.ha-1 kg yield. kg-1 N Mg.ha-1 kg N.ha-1 %

C :N ratio (CN)

Significance *1 * ns ns ns

16 43,6 b2 130 c 9 130 34

17 42,4 ab 123 bc 8 119 27

22 39,3 a 89 ab 8 118 27

24 39,9 a 95 a 8 112 14


Significance *** ns ** ** ns

0 (Control) 30,8 a - 6 a 90 a -

57 35,8 ab 119 7 ab 102 a 22

113 40,5 b 102 8 bc 113 ab 21

170 47,5 c 109 9 c 145 b 33

Linear *** ns *** *** ns

CN*R ns ns ns ns ns 1 *, ** and *** refer to p-values < 0.05, < 0.01 and < 0.001, respectively; ns = not significant.


0

20

40

60

80

100

120

140

160

180

0 50 100 150 200

Tu

be

r n

itro

ge

n a

ccu

mu

lati

on

(kg

N.h

a-1

)



29

4) Nitrogen dynamics: tuber apparent N recovery and N released from silage

a. Tuber apparent nitrogen recovery

C:N ratio did not have a clear effect on tuber ANR. However, ANR was somewhat higher for lower

C:N ratios, and the difference in N recovery can be seen especially between CN16 and CN24 (Table 8

and Figure 13a).

Fertilization rate did not have any significant effect on tuber ANR (Table 8). However, ANR in tubers

was somewhat higher for the highest rate as compared to the lowest and medium rate, which is

contrary to what was expected (Fig. 13b). On one hand, ANR would typically decrease with increasing

N application rate, especially for above-optimal N rates (Larkin and Honeycutt, 2012). According to

Vos (2009), in the case of potatoes (as opposed to cereals and grasses) there is no initial constant

value of ANR followed by a decrease when N supply exceeds the uptake capacities of the crop, but a

continuous decline for every additional unit of N supplied. On the other hand, those results come in

line with previous experiments using the same application rates: fertilization rate did not have an

effect on ANR, however ANR values would increase with increasing rates (Litsos, 2015).

Thus, Figure 14 shows that the highest ANR in tubers was achieved from the treatment with lowest

C:N ratio and highest rate (i.e. CN16-R170). The figure also shows that standard errors are quite high.

Over the whole range of application treatments, 25% of N was recovered in the tubers from the N

applied. Rodrigues et al (1999) also found particularly low values of ANR in potato tubers when

applying organic amendments to the soil. This low efficiency can be attributed to the limited and

shallow root system of the crop. (Stalham and Allen, 2001 in Cambouris et al, 2008). According to

Greenwood and Draycott (1988), due to the low root densities, some of the N applied would be

remoted from the roots for a certain time before being absorbed by the roots.

0

5

10

15

20

25

30

35

40

45

15 17 19 21 23 25

Tu

be

r a

pp

are

nt

nit

rog

en

reco

ve

ry (

%)

C:N ratio

0

5

10

15

20

25

30

35

40

0 50 100 150 200Tu

be

r a

pp

are

nt

nit

rog

en

re

cov

ery

(%)


Figure 13. Tuber apparent nitrogen recovery as affected by C:N ratio (a) and N fertilization rate (b).

Error bars stand for standard errors.

a) b)


30

Figure 14. Tuber apparent N recovery as affected by combined treatments of C:N ratio and fertilization rate.

C:N ratio levels: 16; 17; 22; 24. Fertilization rate levels: 0; 57; 113; 170 kg N ha-1

. Error bars stand for standard

errors.

b. Litterbag experiment

Concerning N released from the silage buried in litterbags, Figure 16 shows that over the 15 week-

period considered there was no significant difference in N release between the C:N ratios. The

maximum amount of N released was reached at 4 weeks after burying the litterbags and was on

average 70%, with CN16 releasing 10% more nitrogen and CN24 releasing 10% less. Values obtained

for CN17 in the 2 first weeks after burying must have been due to contamination or errors in

measurement, as there were no other results among the other measurements that showed a slower

pattern of N release during plant growth compared with the other silages.

In fact, decomposition of plant material depends on C:N ratio of the decomposing material and C:N

ratio of the microbial community in the soil. Fungi and bacteria have different nutrients composition.

In general, C:N ratio of bacteria is expected to be ~3-6 while C:N ratio of fungi approximates ~5-15,

meaning bacteria have a greater C:N ratio than fungi on average (McGill et al., 1981). Therefore,

fungal community is expected to have lower N requirements than bacterial community, so that for

equivalent access to C, there should be a shift to fungal dominance when N is limiting while a shift

towards bacterial dominance should be observed if N is not limited (Hu et al., 2001). Also, when plant

material recalcitrance increases then fungal role is expected to increase. This is related to fungi’s

abilities to degrade lignin which might not have the bacteria (de Boer et al., 2005). Actually, during

the first 4 excavations of litterbags, consequent fungal hyphae were observed, first around the

litterbags and then inside the litterbags indicating there was an active fungi network, which in turn

was not observed during the next excavations. Therefore, there might have been a fungal:bacterial

dominance in the first weeks of decomposition when plant material was quite recalcitrant. However,

it is not known whether this phenomenon was similar or different in the field experiment. Also,

whether the fungal: bacterial dominance can affect the decomposition of plant material is not well

documented yet (Strickland and Rousk, 2010).

Soil microbial activity and subsequent decomposition of plant material causing N release is largely

dependent on climatic factors such as temperature and precipitation, as well as soil properties such

as soil moisture and soil total N and organic C (MacDonald et al, 1995 in Walley). Silage applied at

planting was not subjected to the same climatic conditions as silage buried within litterbags 3 weeks

after planting. Therefore, it is not possible to use the N release patterns observed from

-30,0

-20,0

-10,0

0,0

10,0

20,0

30,0

40,0

50,0

60,0

16 17 22 24 16 17 22 24 16 17 22 24

57 113 170

Tu

be

r a

pp

are

nt

nit

rog

en

re

cov

ery

(%

)

Treatments


31

decomposition of silage in the litterbags as an estimator of N release patterns from the silage applied

at planting in the field experiment. In fact, silage applied earlier in the season has been exposed to

lower temperatures than silage buried in the litterbags. This might have slowed down the microbial

activity therefore causing N immobilization, which was not observed in litterbags due to exposure in

the first weeks to higher temperatures.

However, one can compare the maximum amount of N released with the amount of N recovered in

the tubers. Whereas 70% of N was released from silage, only 25% of N was recovered on average by

the potato crop. Therefore, the ANR in the experiment is considerably low compared to the amount

of N released by the silage in the litterbag experiment. This can be attributed to several reasons.

Firstly, the amount of N released from the litterbag experiment refers to total N, so that the fraction

of mineral N that could be available for the plant out of this total N in unknown. Secondly, silage in

the litterbag experiment was optimally positioned in the ridge horizontally and at a depth of 15 cm.

Silage in the field experiment, however, was incorporated through ecoploughing, which implies it

might not be placed exactly at the depth the potato seeds were planted. Thirdly, the nitrogen

application rate in the litterbags was much lower than in the field experiment. Finally, silage

application distribution in the field experiment was not even as it was broadcasted by hand. There

might be clods of silage mixed in the soil that would take longer time to be decomposed. Shah et al

(2012) who conducted similar experiments using cattle manure on field and in litterbags obtained

similar results and gave similar explanations.

Figure 15. Nitrogen release over time (in weeks after burying) from decomposition of silage in litterbag

experiment of C:N ratio 16; 17; 22; 24.

0

10

20

30

40

50

60

70

80

90

0 2 4 6 8 10 12 14 16

Am

ou

nt

of

nit

rog

en

re

lea

sed

(%

)

Weeks after burying

16

17

22

24


32

IV- Conclusions

The goal of this current research was to investigate the influence of C:N ratio and nitrogen

application rate in grass-clover silage as cut-and-carry fertilizer on organic potato growth and yield.

C:N ratio had a significant effect on the fresh potato yield, with a higher yield in case of C:N ratio 16.

However, decreasing C:N ratio in silage resulted in a yield increase of 10% only, so that it might not

be worthwhile delaying time of harvest of pastures to October-November.

Use of C:N ratio above 20 resulted in lower growth performances in the early stage of canopy

growth, which can be attributed to N immobilization. There was no significant effect of C:N ratio on

tubers N accumulation and apparent N recovery at harvest. However, there was a trend to

decreasing values with increasing C:N ratio. Accordingly, it would be recommended to make use of

late-season potato varieties with such high C:N ratio to ensure optimum use of N from the grass-

clover silage.

Potato canopy light interception as affected by C:N ratio showed some promising results but falling

plants due to shallow ridges disturbed the measurements. Therefore, further research could be done

to reiterate the method used for measuring light interception on potato foliage.

In terms of fertilization rate, the yield difference among 57; 113 and 170 kg N.ha-1 was significant

with a highly positive and strict linear trend. Potato growth performance was improved with

increasing rates, and the amount of N accumulated in the tubers increased as well. Therefore, there

is room for higher yield at higher N application rates. However, this would imply incorporating bigger

amounts of grass-clover silage to the soil, which might be more difficult to handle at application,

especially when ensiled grass-clover has a low DM content. Also, potential risks of N losses in the

environment should be taken into consideration. There was no effect of fertilization rate on tuber

agronomic efficiency and N recovery. However in case of ANR, the value was somewhat higher with

the highest rate, which is opposite to what is generally stated in the literature. Also, the highest

tuber ANR of 43% was reached with lowest C:N ratio and highest fertilization rate. Generally, potato

is a crop that is strongly affected by nitrogen rates, so that fertilization rate should always be taken

into consideration from an economic and environmental point of view.

Based on the litterbags experiment, C:N ratio of silage had no effect on N release pattern. There was

a high difference between the amount of total N released during decomposition in the litterbags and

the amount of N recovered in the tubers. However, the potential fraction of plant-available N in the

total N released from the litterbags was not known. Therefore, further research is needed regarding

availability of mineral N in the soil from grass-clover silage.

Based on the results of the current study, grass-clover silage can be recommended as a suitable

substituent to cattle manure as it provided a yield of 41 ton.ha-1 on average. Conflicting results with

general literature were found with the effect of fertilization rate on fresh tuber yield and apparent N

recovery in the tubers, so that further research should be conducted on the combined effect of C:N

ratio in grass-clover silage and N application rate on potato yield components.


33

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Pauline Martel Thesis Proposal

37

VI- Appendices

Appendix 1. Results of the 3 chemical analysis of grass-clover silage for C:N ratio determination

Table 1. Silage characteristics from the 1st analysis – 11 march 2015

Silage # Harvest time DM N P K NH4-N NO3-N Total Nmin PO4-P K20-K C OM ash C:N

% g/kg %

1 May 76 15,6 - - - -

- - 418,40 90,9 9 27

2 June-July 60 22,1 - - - -

- - 432,50 90,7 9 20

3 Aug-Sept 45 28,3 - - - -

- - 440,40 89,6 10 16

4 Oct-Nov 16 31,1 - - - - - - 409,10 79,6 20 13

Table 2. Silage characteristics from the 2nd analysis – 16 april 2015

Silage # Harvest time DM N P K NH4-N NO3-N Total Nmin PO4-P K20-K C OM ash C:N pH-H2O

% -------------------------------------------- g/kg ------------------------------------------------------------- ------%--------

1 May 69 17,4 3,6 30,9 0,65 0,00 0,66 3,23 30,71 450,2 90,8 9,2 26 5,7

2 June-July 69 16,7 3,4 30,5 0,68 0,00 0,68 2,88 35,55 454,2 90,7 9,3 27 5,4

3 Aug-Sept 50 25,5 4,4 35,8 0,79 0,01 0,80 4,09 36,95 494,1 88,3 11,7 19 4,9

4 Oct-Nov 21 23,4 4,4 24,5 0,65 0,00 0,65 3,73 34,04 444,9 76,1 23,9 19 4,5

Table 3. Silage characteristics from the 3 analysis – 8 may 2015

Silage # Harvest time DM N P K NH4-N NO3-N Total Nmin PO4-P K20-K C OM ash C:N pH-H2O

% -------------------------------------------- g/kg --------------------------------------------------- ------%-------

1 May - 20,5 - - - - - - - 439,4 - - 21 -

2 June-July - 20,2 - - - - - - - 438,7 - - 22 -

3 Aug-Sept - 27,1 - - - - - - - 445,0 - - 16 -

4 Oct-Nov - 24,4 - - - - - - - 382,3 - - 16 -


38

Appendix 2. Experimental data

Table 9. Experimental data of emergence date, plant height, canopy volume and light interception.

Block C:N

ratio

Fertilization

rate

Emergence

date

Plant height Canopy volume Light interception

7 WAP 9 WAP 11 WAP 7 WAP 9 WAP 11 WAP 7 WAP 9 WAP 11 WAP

kg N ha-1

DAP ---------------cm------------------ --------------------cm3-------------------- ----------------%-------------------

1 24 57 42 13 50 62 2 446 13 973 83 101 5,6 52,2 84,5

1 24 113 41 13 55 68 2 287 15 956 138 679 26,4 72,2 90,2

1 24 170 36 21 58 67 6 278 25 617 102 458 19,4 66,6 90,6

1 22 57 40 18 62 69 4 694 26 280 96 330 16,8 58,3 88,3

1 22 113 40 20 57 68 5 643 23 814 92 461 12,4 71,1 90,4

1 22 170 42 14 55 68 3 316 21 720 82 318 31,8 73,7 79,7

1 17 57 40 27 62 70 12 326 42 051 126 699 20,7 61,5 83,2

1 17 113 41 21 60 66 6 451 35 111 58 443 14,3 60,0 78,0

1 17 170 42 16 54 71 4 329 20 552 140 994 27,4 69,9 82,7

1 16 57 41 16 57 69 5 021 23 288 100 628 14,1 60,2 83,2

1 16 113 41 21 55 58 5 949 29 271 89 420 13,3 61,7 87,5

1 16 170 38 20 63 70 6 148 30 845 162 652 16,5 65,8 80,1

1 C 0 42 17 52 62 4 837 16 936 67 532 4,3 43,9 69,9

2 24 57 40 21 60 62 6 806 56 316 74 493 20,4 73,4 87,3

2 24 113 38 19 56 70 4 863 69 253 107 377 15,0 76,0 90,7

2 24 170 37 25 50 77 13 783 62 354 126 653 25,7 80,0 90,1

2 22 57 39 22 57 69 8 310 79 448 110 627 2,4 73,6 82,6

2 22 113 39 17 63 79 4 534 70 398 103 497 18,7 64,8 87,4

2 22 170 40 19 55 72 5 367 47 410 113 615 20,0 61,0 80,9

2 17 57 40 21 66 71 7 894 119 707 99 803 33,1 70,5 90,2

2 17 113 38 26 63 74 10 705 80 625 145 441 14,3 67,9 90,9

2 17 170 41 18 59 75 6 275 68 537 93 108 21,7 70,2 89,2

2 16 57 39 19 60 69 4 872 97 937 131 904 25,7 65,2 84,7

2 16 113 39 20 62 70 7 015 103 234 91 513 2,1 69,4 88,4

2 16 170 38 25 65 71 13 019 90 391 131 698 26,9 80,5 72,0

2 C 0 37 15 58 65 3 817 63 381 98 499 6,7 69,4 85,5

3 24 57 39 22 56 65 7 862 21 465 81 400 17,3 67,3 82,4

3 24 113 40 20 57 64 6 584 25 266 99 752 21,2 61,6 80,0

3 24 170 37 26 63 75 12 452 35 134 117 179 31,7 72,7 79,5

3 22 57 40 14 52 68 3 378 22 346 115 488 25,2 58,9 87,8

3 22 113 41 20 54 59 6 877 21 200 67 633 14,2 59,7 85,7

3 22 170 40 24 57 73 9 307 21 530 120 371 17,2 67,7 85,8

3 17 57 40 23 59 71 10 426 23 103 68 658 18,4 70,8 82,5

3 17 113 36 23 64 67 8 394 39 441 85 278 39,1 73,4 88,9

3 17 170 39 23 62 73 9 487 33 021 120 830 26,7 72,5 81,3

3 16 57 40 23 58 67 9 343 26 443 72 858 14,0 69,5 79,0

3 16 113 40 25 63 73 11 633 31 358 93 541 31,1 67,9 69,5

3 16 170 36 30 67 76 14 693 47 521 163 745 23,0 82,6 80,8

3 C 0 40 23 55 64 10 512 14 825 80 023 33,5 64,4 79,2

4 24 57 37 29 51 57 11 261 50 581 60 305 27,4 41,9 75,4

4 24 113 41 20 50 64 6 061 51 259 91 897 11,3 72,9 87,5


39

4 24 170 41 22 50 64 8 333 53 694 84 335 23,3 76,7 86,1

4 22 57 41 20 54 57 5 508 57 436 75 819 19,2 75,6 88,2

4 22 113 41 21 51 66 7 565 57 679 94 552 19,7 54,8 87,6

4 22 170 40 22 59 69 7 662 102 906 108 421 15,8 72,5 80,4

4 17 57 41 18 56 68 5 513 57 940 82 495 14,6 72,7 75,8

4 17 113 41 28 61 64 11 411 76 390 82 769 17,3 78,5 84,1

4 17 170 37 19 64 67 6 402 86 779 104 583 16,7 78,2 87,6

4 16 57 39 25 56 68 9 508 75 288 113 382 20,6 66,4 78,5

4 16 113 41 25 65 63 12 473 108 449 76 050 25,0 62,7 78,9

4 16 170 40 16 60 62 4 435 85 316 112 410 19,9 74,8 80,9

4 C 0 38 21 52 62 7 591 59 577 71 054 30,1 74,7 82,3


40

Table 10. Experimental data of fresh tuber yield, dry matter yield, agronomic nitrogen efficiency, tuber nitrogen

accumulation and tuber apparent nitrogen recovery.

Block C:N

ratio Fertilization rate Fresh yield DM yield AE

N

accumulation ANR

kg N ha-1 ton ha-1 Mg ha-1 kg yield kg-1 N kg N ha-1 %

1 24 57 34,3 6,9 92,3 108,7 34,2

1 24 113 32,8 6,4 33,0 123,7 30,6

1 24 170 47,9 9,0 110,9 139,6 29,7

1 22 57 35,4 7,7 111,6 106,9 31,2

1 22 113 32,7 6,3 32,1 89,5 0,2

1 22 170 40,4 7,9 66,9 148,9 35,2

1 17 57 37,1 7,5 141,8 110,4 37,2

1 17 113 40,0 8,0 96,3 116,0 23,7

1 17 170 42,9 7,7 81,5 145,5 33,1

1 16 57 36,4 7,0 128,6 102,9 24,1

1 16 113 42,9 8,7 122,5 127,2 33,6

1 16 170 49,2 8,6 118,5 194,8 62,1

1 C 0 23,6 5,0 - 67,3 -

2 24 57 32,8 6,4 65,4 76,6 -22,0

2 24 113 43,5 8,4 127,5 133,6 39,3

2 24 170 52,4 10,8 137,2 188,3 58,3

2 22 57 32,6 6,2 62,4 106,7 30,8

2 22 113 43,7 7,8 129,3 141,5 46,3

2 22 170 46,9 8,8 104,8 132,6 25,5

2 17 57 40,4 8,3 199,2 118,5 51,4

2 17 113 44,2 8,9 133,9 123,5 30,4

2 17 170 56,8 10,6 163,0 170,9 48,1

2 16 57 38,6 8,2 166,4 121,0 55,8

2 16 113 42,9 8,2 122,5 97,7 7,6

2 16 170 50,7 10,1 127,5 154,6 38,5

2 C 0 32,0 5,0 - 78,6 -

3 24 57 34,1 7,1 88,9 74,5 -25,7

3 24 113 43,6 8,9 129,0 104,3 13,4

3 24 170 46,4 8,8 101,7 145,8 33,3

3 22 57 35,3 6,9 109,7 113,3 42,3

3 22 113 40,1 7,7 97,6 131,4 37,4

3 22 170 41,0 7,3 70,4 100,8 6,8

3 17 57 36,8 7,3 136,3 113,0 41,8

3 17 113 41,5 7,4 110,3 107,7 16,4

3 17 170 46,1 8,5 100,2 127,5 22,5

3 16 57 33,9 6,5 85,1 85,7 -6,0

3 16 113 51,1 10,3 194,8 127,7 34,1

3 16 170 53,5 10,0 143,8 159,0 41,1

3 C 0 34,7 6,9 - 111,6 -

4 24 57 38,4 7,8 164,5 83,3 -10,4

4 24 113 32,4 6,4 29,1 79,5 -8,6

4 24 170 40,0 7,8 64,1 81,8 -4,3

4 22 57 32,4 6,8 59,1 102,3 22,9

4 22 113 40,5 8,4 101,5 101,2 10,7


41

4 22 170 50,3 9,5 124,7 140,5 30,2

4 17 57 36,1 7,3 123,7 92,9 6,6

4 17 113 37,9 7,7 78,5 78,5 -9,5

4 17 170 48,6 9,8 115,1 127,8 22,7

4 16 57 38,4 7,9 164,2 112,3 40,6

4 16 113 38,7 7,4 85,6 124,8 31,5

4 16 170 47,0 9,2 105,5 163,1 43,5

4 C 0 31,5 6,5 - 101,9 -

Table 3. Experimental data of total N released in litterbags experiment

Time Block C:N ratio Ntot release

WAB

%

1 1 24 37,28

1 1 22 42,45

1 1 17 6,00

1 1 16 45,07

1 2 24 33,31

1 2 22 51,85

1 2 17 -

1 2 16 46,47

1 3 24 18,50

1 3 22 38,53

1 3 17 27,30

1 3 16 42,00

1 4 24 32,40

1 4 22 46,14

1 4 17 -

1 4 16 32,67

2 1 24 30,47

2 1 22 47,62

2 1 17 30,02

2 1 16 43,02

2 2 24 38,18

2 2 22 51,45

2 2 17 24,31

2 2 16 56,64

2 3 24 33,65

2 3 22 48,31

2 3 17 21,87

2 3 16 63,18

2 4 24 53,72

2 4 22 49,50

2 4 17 30,80

2 4 16 48,51

4 1 24 61,17

4 1 22 74,35

4 1 17 75,72

4 1 16 84,00


42

4 2 24 58,24

4 2 22 66,98

4 2 17 69,59

4 2 16 78,91

4 3 24 63,70

4 3 22 68,32

4 3 17 69,75

4 3 16 74,55

4 4 24 63,54

4 4 22 71,16

4 4 17 56,33

4 4 16 84,47

8 1 24 59,40

8 1 22 70,70

8 1 17 53,18

8 1 16 57,26

8 2 24 55,56

8 2 22 62,82

8 2 17 57,32

8 2 16 66,46

8 3 24 61,17

8 3 22 70,96

8 3 17 -

8 3 16 78,27

8 4 24 70,03

8 4 22 68,81

8 4 17 59,90

8 4 16 68,63

12 1 24 70,98

12 1 22 69,54

12 1 17 53,60

12 1 16 59,67

12 2 24 60,58

12 2 22 73,04

12 2 17 72,80

12 2 16 69,13

12 3 24 58,99

12 3 22 60,61

12 3 17 61,98

12 3 16 70,94

12 4 24 58,44

12 4 22 72,70

12 4 17 51,39

12 4 16 64,02

15 1 24 74,21

15 1 22 74,92

15 1 17 68,19

15 1 16 68,69

15 2 24 66,06


43

15 2 22 66,96

15 2 17 78,74

15 2 16 72,20

15 3 24 64,47

15 3 22 75,82

15 3 17 61,03

15 3 16 58,90

15 4 24 70,03

15 4 22 68,45

15 4 17 54,74

15 4 16 61,18

grass-clover silage as cut-and-carry fertilizer in organic

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