The ability of the Milpa growth system to reduce the ...library.au.dk/fileadmin/ · The maize, bean...

The ability of the Milpa growth system to reduce the negative

impacts by Phyllophaga vetula on maize, with the influence of

arbuscular mycorrhiza fungi and fertilizer on the maize-

Phyllophaga vetula interaction.

INTERACTIONS Phyllophaga vetula Arbuscular mycorrhiza fungi

and fertilizer

Mikkel Møller Jørgensen

Agrobiology – Plant Nutrition and Health

2nd

of June 2017

Growth systems

Side 1 af 60

Title: The ability of the Milpa growth system to reduce the negative

impacts by Phyllophaga vetula on maize, with the influence of

arbuscular mycorrhiza fungi and fertilizer on the maize-

Phyllophaga vetula interaction.

Author: Mikkel Møller Jørgensen

Student registration

number:

201205358

Master’s program: Agrobiology – Plant Nutrition and Health

Aarhus University

Credits of the thesis: 60 ECTS

Date of submission:

Date of examination:

2nd

of June 2017

16th

of June 2017

Supervisors: Main supervisor:

Associate Professor Sabine Ravnskov

Department of Agroecology – Crop Health, AU

Co-supervisor:

Investigador Titular C John Larsen (Associate Professor)

Instituto de investigaciones en ecosistemas y sustentabilidad

(IIES), UNAM Morelia

Faculty Science and Technology

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Preface

This thesis is submitted in partial fulfilment of the requirements for the Master degree (MS) in

Agrobiology – Plant Nutrition and Health at the faculty of Science and Technology, Aarhus

University, Denmark. This project lasted for 11 months and was part of a larger project,

“Importance of beneficial microbes in the rhizosphere in the sustainable production of maize”,

by CONACYT-2102-179319. The project was split in two, where six months were used for

the experimental part in Mexico, four months were used for the writing process and the last

month preparing the defence of the thesis.

First of all, I would like to thank my supervisor, Sabine Ravnskov, for giving me the

opportunity to conduct this research and contact to John Larsen and for her excellent

supervision and advices through my writing process. Secondly, I would like to thank my co-

supervisor, John Larsen, for making the stay in Mexico as easy and eventful as possible for

Rikke and me, which meant a great lot for the both of us. I would also thank him for the

supervision, help and advices through the experimental and post-experimental work of my

thesis. Thirdly, I would like to thank Rikke, my girlfriend, for coming along with me to

Mexico, where she supported me through the six months in Mexico and the final months in

Denmark, and furthermore, was able to share this experience with me, which meant a lot.

Fourthly, I would like to thank the laboratory of Agroecología at the department;

Investigaciones en Ecosistemas y Sustentabilidad (IIES) at Universidad Nacional Autónoma

de Mexico, Campus Morelia (UNAM, Morelia), for inviting me into the laboratory and the

Mexican culture, including helping with harvesting of the pot experiments. Further, thanks to

Miguel Bernardo Najera-Rincon for helping with collecting the Phyllophaga vetula larvae and

observing them through the quarantine period. A thank you also goes to my parents who,

during their visit in Mexico, helped harvest one of the pot experiments. Finally, a thank you

goes to the scholarships of Kølpin Ravns Legat, Fonden Frands Christian Frantsens Legat and

Agronomfonden, who financially supported my stay in Mexico, so the collection of data to

my master project was possible.

_______________________________________________

Date Mikkel Møller Jørgensen

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Table of Content

Preface ........................................................................................................................................ 2

Table of Content ......................................................................................................................... 3

List of figures ............................................................................................................................. 6

List of tables ............................................................................................................................... 6

List of pictures ............................................................................................................................ 6

Abstract ...................................................................................................................................... 7

Abstrakt ...................................................................................................................................... 8

Summary .................................................................................................................................... 9

1 Introduction ...................................................................................................................... 11

1.1 Plants ......................................................................................................................... 11

1.1.1 Maize .................................................................................................................. 11

1.1.2 Bean .................................................................................................................... 12

1.1.3 Squash ................................................................................................................ 12

1.2 Growth systems ......................................................................................................... 13

1.2.1 Milpa .................................................................................................................. 13

1.2.2 Monoculture ....................................................................................................... 14

1.3 Phyllophaga vetula .................................................................................................... 15

1.4 Arbuscular Mycorrhiza Fungi ................................................................................... 16

1.5 Organic and inorganic fertilization of plants ............................................................. 16

1.6 Interactions ................................................................................................................ 17

1.7 Hypothesis ................................................................................................................. 20

1.8 Objective .................................................................................................................... 20

1.9 Research questions .................................................................................................... 20

2 Material and Method ........................................................................................................ 21

2.1 Organisms .................................................................................................................. 21

2.1.1 Plants .................................................................................................................. 21

2.1.2 Phyllophaga vetula ............................................................................................. 21

2.1.3 Arbuscular mycorrhiza fungi ............................................................................. 22

2.2 Experimental designs ................................................................................................. 22

2.2.1 Experiment 1 ...................................................................................................... 22

2.2.2 Experiment 2 ...................................................................................................... 22

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2.2.3 Experiment 3 ...................................................................................................... 23

2.2.4 Experiment 4 ...................................................................................................... 23

2.3 Experimental setup .................................................................................................... 25

2.3.1 Soil and sand ...................................................................................................... 25

2.3.2 Pots preparation .................................................................................................. 25

2.3.3 Nutrient solutions and fertilization ..................................................................... 26

2.3.4 Sowing ................................................................................................................ 27

2.4 Growth conditions ..................................................................................................... 27

2.4.1 Climatic conditions ............................................................................................ 27

2.4.2 Reallocation and removing of plants .................................................................. 28

2.4.3 Adding of P. vetula ............................................................................................ 28

2.4.4 Extra nutrients .................................................................................................... 29

2.4.5 Unwanted pest .................................................................................................... 29

2.5 Harvest and end of experiments ................................................................................ 30

2.6 Analyzes .................................................................................................................... 30

2.6.1 Biomass of plant shoot ....................................................................................... 30

2.6.2 Biomass of plant root ......................................................................................... 30

2.6.3 Growth of Phyllophaga vetula ............................................................................ 31

2.6.4 Arbuscular mycorrhiza colonization .................................................................. 31

2.6.5 Statistical Analysis ............................................................................................. 32

3 Results .............................................................................................................................. 33

3.1 Experiment 1.............................................................................................................. 33

3.1.1 Biomass of maize plants ..................................................................................... 33

3.1.2 AMF colonization of maize roots ....................................................................... 35

3.1.3 Biomass and survival of P. vetula larvae ........................................................... 36

3.1.4 Biomass of total plants ....................................................................................... 36

3.1.5 AMF colonization of total roots ......................................................................... 38

3.2 Experiment 2.............................................................................................................. 39

3.2.1 Biomass of plants ............................................................................................... 39

3.2.2 AMF colonization of maize, bean and squash roots .......................................... 41


3.3 Experiment 4.............................................................................................................. 42

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3.3.1 Biomass of P. vetula larvae ................................................................................ 42


3.4 Experiment 3.............................................................................................................. 44

3.4.1 Biomass of plants ............................................................................................... 44



4 Discussion ........................................................................................................................ 46

4.1 Growth systems’ effect on maize .............................................................................. 47

4.2 Maize plant herbivory by P. vetula ........................................................................... 47

4.3 AMF colonization in roots of Milpa or monoculture growth systems ...................... 48

4.4 Herbivory by P. vetula on AMF colonized maize roots ............................................ 49

4.5 AMF and fertilizer impacts on P. vetula ................................................................... 50

4.5.1 Biomass .............................................................................................................. 50

4.5.2 Length ................................................................................................................. 51

5 Conclusion ........................................................................................................................ 52

6 Perspectives ...................................................................................................................... 53

7 Bibliography ..................................................................................................................... 55

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List of figures Figure 1. The order of soil and sand applied to the pots in exp. 1, 2 and 3 .................................. 25

Figure 2. Diagram of the average biomass of the maize shoots and roots in experiment 1 ........... 33

Figure 3. Average AMF colonization percentage of maize roots in experiment 1 ....................... 35

Figure 4. Total biomass of the shoots, roots and AMF colonization in experiment 1 .................. 36

Figure 5. Total AMF colonization percentage of roots in experiment 1 ...................................... 38

Figure 6. The biomass of shoots and roots from maize, bean and squash in experiment 2 ........... 39

Figure 7. AMF colonization of roots from maize, bean and squash in experiment 2 ................... 41

List of tables Table 1. The experimental design of exp. 1 ............................................................................. 22

Table 2. The experimental design of exp. 2 ............................................................................. 23

Table 3. The experimental design of exp. 3 ............................................................................. 23

Table 4. The experimental design of exp. 4. ............................................................................ 24

Table 5. The nutrients solutions used in the experiments ........................................................ 26

Table 6. The effect of Growth system and P. vetula on average maize shoots, roots and AMF

colonization in experiment 1. ................................................................................................... 34

Table 7. Biomass, biomass gain and survival percentage of P. vetula in experiment 1. ......... 36

Table 8. The effect of Growth system and P. vetula on shoots and roots in experiment 1. ..... 37

Table 9. The effect of Crop and P. vetula on shoot biomass, root biomass and AMF

colonization in experiment 2 .................................................................................................... 39

Table 10. Biomass, biomass gain and survival percentage of P. vetula in experiment 2 ........ 42

Table 11. Biomass, biomass gain, length and length gain of P. vetula in experiment 4 .......... 42

Table 12. The effect of AMF and fertilizer on the biomass, biomass gain length and length

gain P. vetula in experiment 4 .................................................................................................. 43

Table 13. AMF colonization percentage in maize roots used as feed in experiment 4. ........... 43

Table 14. Biomass of shoots and roots and AMF colonization percentage in experiment 3. .. 44

Table 15. The effect of phosphorous and P. vetula on the biomass of shoots and roots and

AMF colonization percentage in experiment 3 ........................................................................ 44

Table 16. Biomass, biomass gain and survival percentage of P. vetula in experiment 3 ........ 45

List of pictures Picture 1. Determining if the larvae were of the P. vetula specie. ........................................... 21

Picture 2. Collecting of the P. vetula larvae in Zacápu with the local farmer. ........................ 21

Picture 3. Net cover for future unwanted pest attacks. ............................................................. 29

Picture 4. The extent of the unwanted pest, where the worst attacked maize plant is shown. . 29

Picture 5. Maize, bean and squash plants from a Milpa growth system pot from exp. 1. ........ 30

Picture 6. The investigated interactions in the present study ................................................... 46

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Abstract The maize, bean and squash plants are an ancient intercropping system (Milpa) and can over

yield due to architectural differences. The root-feeding Phyllophaga vetula (P. vetula) larva

can cause severe economic damages in monoculture maize and the conventional control

method by using chemical pesticides inflict unsustainable effects on the environment. AMF

are plant beneficial rhizosphere microorganisms which increase nutrient uptake in plants. It is

hypothesized that Milpa can reduce the negative impact by P. vetula on maize. Further, a

change in the biotic (arbuscular mycorrhiza fungi) and abiotic (Fertilizer) soil environments

influences the maize-P. vetula interaction. The interactions were investigated in pot

experiments in a greenhouse with a native arbuscular mycorrhiza fungi (AMF) population,

Milpa/monoculture growth systems, P. vetula larvae (with and without), growth of the

individual Milpa plant species and a small study of the AMF-fertilizer-P. vetula interactions.

The Milpa growth system did not enhance the biomass growth of maize, but reduced the

biomass growth of the maize plant. Furthermore, the biomass reduction in maize shoots of P.

vetula root herbivory increased in the Milpa growth system, by a 22.22% reduction compared

to the monoculture growth system with a 10.78% reduction. AMF did not influence the P.

vetula biomass and fertilizer did influence the biomass. In conclusion the Milpa growth

system did not reduce the negative impact by P. vetula on maize but enhanced the biomass

reduction of the Milpa growth system compared to the monoculture. The growth parameters

in P. vetula did not express any affect either in the Milpa growth system or in the presence of

AMF.

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Abstrakt

Majs, bønne og squash er et gammelt samdyrkningssystem (Milpa) og har vist at kunne øge

udbyttet på de involverede plantearter. Den rod-spisende P. vetula larve kan give store

økonomiske skader på majs i monokulturer, mens den konventionelle bekæmpelses metode

med kemisk fremstillede pesticider påfører ubæredygtige virkninger på miljøet. AMS er

plante gunstige mikroorganismer, som er i stand til at øge næringsstofoptaget i planter.

Studiets hypotese lyder at Milpa kan reducere de negative påvirkninger P. vetula har på majs.

Yderligere vil en ændring af de biotiske (Arbuskulær mykorrhiza svampe) og abiotiske

(Gødning) jord miljøer influere på majs-P. vetula interaktionen. Undersøgelserne har fundet

sted med potteeksperimenter i et drivhus med en naturlige arbuskulær mykorrhiza svamp

(AMS) population, Milpa/monokultur systemer, tilstedeværelsen af P. vetula larver (med og

uden), individuel vækst af Milpa planterne og et lille studie til at belyse AMF-gødning-P.

vetula interaktionerne. Milpa systemet kunne ikke øge skudvæksten på majs, men reducerede

den derimod. Derudover blev skudvæksten i majs yderlig reduceret af rod herbivory af P.

vetula, hvilket gav til sammenligning en reduktion på 22.22% i Milpa majs og 10.78% i

monokultur majs. AMF ikke influerede biomassen i P. vetula larvaer mens gødnings typen

influerede biomassen. Som konklusion kan det siges, at Milpa systemet ikke kan øge væksten

i majs, men derimod reducerer den sammenlignet med monokultur systemet. Derudover

kunne Milpa system ikke reducerer de negative følger af rod herbivory forårsaget af P. vetula

larverne. Vækst faktorerne i P. vetula blev ikke påvirket af hverken Milpa systemet eller

tilstedeværelsen af AMS.

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Summary

Maize, bean and squash plants are part of an ancient intercropping system (Milpa) and can

over yield due to architectural differences. The complimentary architectural structures of the

plant species in the Milpa growth system should enhance the growth in all three plant species.

The growth enhancement of the plant species is influenced by an increase in nutrient uptake,

with different nutrient uptake strategies, and a suppressing effect towards weeds. The root-

feeding P. vetula larva can cause severe economic damages in monoculture maize and is an

important pest in the Mesoamerican area where it is reported to reduce maize yield up to 50%.

Today the most applied control method, towards the P. vetula larvae, is the use of chemical

pesticides. However, the pesticides do inflict unsustainable effects on the environment, and

therefore alternative sustainable control methods are investigated, as for instance where

intercropping. Other plant beneficial organisms could also function as a control method, such

as AMF. These fungi are reported to increase the nutrient uptake in plants and transfer

nutrients between plants of different species. With the increased nutrient uptake and a transfer

of the nutrient between the plant species, the growth is thought to increase even further to

withstand the feeding pressure by P. vetula. Three experiments were conducted as pot

experiments in a greenhouse at Agroecología at the department of Investigaciones en

Ecosistemas y Sustentabilidad (IIES) at Universidad Nacional Autónoma de Mexico, Campus

Morelia (UNAM, Morelia) as random block designs. A fourth experiment was conducted as a

small complementary cafeteria experiment. The first experiment was conducted with

Milpa/monoculture growth systems and P. vetula (with and without) as treatments, while the

second was conducted with maize, bean or squash and P. vetula (with and without). The third

pot experiment was conducted with phosphorous (with and without) and P. vetula (with and

without), while the factors in the fourth experiment were arbuscular mycorrhiza fungi (AMF)

(with or without) and fertilizer (mineral or organic). The Milpa growth system did not

enhance the biomass growth of maize, but on the contrary reduced the biomass growth of the

maize plant which is in contrast to the literature. The different results in this study compared

to the literature could be due to the soil volumes used. Furthermore, the herbivory effect by P.

vetula on maize did increase in the Milpa growth system compared to the monoculture growth

system, which could be a prolonged effect of the reduced biomass of maize in the Milpa

growth system. The presence of AMF was not able to reduce the biomass of P. vetula, but the

AMF colonization percentage was, however, negatively affected by the presence of P. vetula,

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which could lead to the idea that P. vetula prefer feeding on the AMF colonization points of

the roots. In conclusion the results showed that the Milpa growth system reduced the biomass

of maize, and further, was not able to reduce the negative impacts on maize by P. vetula, but

on the contrary the P. vetula larvae did enhance the negative impacts on the biomass of maize.

Finally AMF could not affect the biomass of P. vetula, but the AMF colonization percentage

was affected by the P. vetula larvae, while the fertilizer type had an influence on the biomass

and length of the larvae.

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1 Introduction

Maize (Zea Mays) originate from the Mesoamerica region and is today one of the most

produced and consumed crops in the world (FAOSTAT, 2015). Through time maize has been

part of both intercropping and monoculture systems, in which evidence suggests that maize

has been a part of an intercropping system called Milpa or the “three sisters”. This

intercropping system consists of maize, bean and squash, and it has existed for thousands of

years (Smith, 2006) even before the rise of the Mayan empire which had its classic period

from around 200 AD to 1000 AD. Since then a more productive way to produce maize has

emerged, and today huge areas solely consisting of maize plants are wide spread all over the

world. This is done to increase food security for the world’s population and to fulfil the needs

of the human population. However, this practice of using monoculture on huge areas has

inflicted an increase in pest attacks, plant diseases etc. because the crops are clones to unify

management (Perez-Garcia and del Castillo, 2016). The P. vetula larva is a common pest in

the Mesoamerica region, from where the maize originated, and is reported to attack a broad

range of crop roots with economic losses in maize as a consequence (Jackson and Klein,

2006). There is often used chemical fertilizer to control P. vetula in maize (Miah et al., 1986,

Shenefelt and Simkover, 1951). With the recent awareness for the more harmful effects of

pesticides on the surrounding environments and human health (Gomez-Arroyo et al., 2011)

alternative ways to control the P. vetula larvae and other pests are investigated. Therefore it

could be interesting to take a look back on the intercropping system Milpa as a way to control

the P. vetula larva. Maize form a mutualistic symbiosis with AMF, and the symbiosis is

known to alter plant growth and health (Smith and Smith, 2011b). The formation of AM

symbiosis has been shown to increase nutrient uptake and a content of defence compounds,

which can have an effect on herbivory (Bennett et al., 2006).

1.1 Plants

1.1.1 Maize

Maize (Zea Mays) originate from Mesoamerica, and is part of the Poaceae family, but it is

today distributed to the world as an important crop alongside wheat (FAOSTAT, 2015).

Maize is used for several purposes such as human food, animal feed, biofuels etc. Maize is

also produced in Denmark, and compared to Mexico there is often used a short season maize

in Denmark (Nielsen et al., 2015), because of the relative short growth season. The potential

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for maize production in Denmark is measured in Maize Heat Units (MVE) (Nielsen et al.,

2015). Maize do not tolerate temperatures below the freezing point, and the maize growth

season in Denmark runs from mid of April to mid of October (Planteavl, 2003). In 2016,

maize production occupied 176,812 ha which is corresponding to 6.78% of the arable land in

Denmark (Statistik, 2016).

Throughout the growth season the maize plant will develop a root system till a depth of 50 to

100 cm, and the main part of the roots is located in the upper layers and has the highest

density just below the soil surface (Hund et al., 2009, Postma and Lynch, 2012). The leaves

however can grow to a height of 4.5 m (Postma and Lynch, 2012).

1.1.2 Bean

Beans (Phaseolus vulgaris) are a genus from the Fabaceae family and originate from North

and South America. The Fabaceae family is also commonly known as legume, and during its

annual growth cycle the bean plants form a symbiosis with the soil bacteria Rhizobium. This

symbiosis produces nodules on the roots wherein the bacteria can live. These nodules are able

to fixate atmospheric nitrogen and therefore the bean plant is independent of the soil nitrogen

level (Saeki, 2011). In Denmark beans do not belong to the most produced crop because they

need a lot of sun. Legumes occupied 15,749 ha in 2016, which is 0.6% of the total arable area

(Statistik, 2016).

The bean root development is similar to maize, however, smaller (Postma and Lynch, 2012).

The development happens in the upper layers of the soil and down to around 50 cm, where the

highest density is just below the soil surface (Postma and Lynch, 2012). The over ground

biomass develops long stalks and tendrils which is used to climb on other plants or elements

in their surroundings as supporting points for their growth towards the sunlight.

1.1.3 Squash

Squash (Curcubita Pepo) is from the Cucurbitaceae family and originate from the

Mesoamerica region, and is today widely distributed as an edible fruit (FAOSTAT, 2014). In

Denmark, squash is hardly produced on a larger scale (FAOSTAT, 2014).

https://en.wikipedia.org/wiki/Cucurbitaceae

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The development of the squash root system is a little different than maize and bean root

systems because the root density of squash increases till 30 cm below the soil surface and

hereafter decreases faster than maize and bean (Postma and Lynch, 2012). Most squash plants

are herbaceous vines that grow several meters in length and produce tendrils, and the shoots

of the plant are big and functions as parasols. In that way they cover a large area and have a

big canopy below their shoots, and the development of weed is at a minimum because of lack

of sunlight (Liebman and Dyck, 1993).

1.2 Growth systems

1.2.1 Milpa

The Milpa Growth system is an intercropping system of maize, bean and squash, and has its

origin in the Mesoamerica region (Smith, 2006). This intercropping growth system is thought

to go back thousands of years (Smith, 2006), and when the rise of the Mayan civilization

came, the classic period was from around 200 AD to 1000 AD, the milpa-system had existed

for some time. Maize, bean and squash have structural and functionally complementarity

(Loreau and Hector, 2001, Weaver and Bruner, 1927) in both shoot and root development

(Postma and Lynch, 2012). The height of the maize can be used as a supporting point for the

climb of the bean plant, while the beans at the same time closes the gaps in the canopy of the

maize. The vines and shoots of the squash plant close the canopy below and by that conserve

water and nutrient in a living mulch (Liebman and Dyck, 1993). This constellation of

intercropping can also enhance weed suppression (Liebman and Dyck, 1993). Below ground

the different root structures (Weaver and Bruner, 1927) enhance the nutrient depletion of the

soil (Dwivedi et al., 2016). The nodules on the bean roots counts as a niche factor to the

nutrient depletion of the soil because it insures atmospheric nitrogen fixation.

Biodiversity is increased in an intercropping system compared to monoculture systems (Isabel

Moreno-Calles et al., 2012, Perez-Garcia and del Castillo, 2016), and the intercropping

system will increase the biodiversity further by attracting associated animals and

microorganisms (Letourneau et al., 2011, Letourneau, 1986), because the number of niches

increases with increased plant diversity. The biodiversity can influence the intercropping

systems on several factors (Dwivedi et al., 2016, Kohl et al., 2014, Loreau and Hector, 2001).

The nutrient uptake can be altered in an intercropping systems and thereafter alter the nutrient

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composition of the plants (Dwivedi et al., 2016, Loreau and Hector, 2001, Vesterager et al.,

2008) which can be controlled by the relation of competition and collaborative (Li et al.,

2014, Stern, 1993). Soil degradation reduces the soils fertility, and studies have shown that

when intercropping with legumes the soil-N enhances (Vesterager et al., 2008). Increased

plant biodiversity can enhance and shift the diversity in other organism groups such as

predators (Song et al., 2012) and beneficial soil organisms (Ekesi et al., 1999, Kohl et al.,

2014). At least intercropping systems can be used as a reservoir for agrobiodiversity to

maintain the genetic diversity of agricultural crops (Perez-Garcia and del Castillo, 2016).

The Milpa growth system is not used in big-scale farming, but rather used in small family

scaled households and communities (Isabel Moreno-Calles et al., 2012, Ortiz-Timoteo et al.,

2014). The reason for this is the multi-cropping system which Milpa is, where 1) all the crops

do not ripen at the same time, 2) the fruits are positioned differently on the different plant

species, 3) the different use of the fruits and plants and 4) the different fruits need different

handling. In other words when the cultivation practice shift towards a more industrialized

management it is difficult to harvest three different plant species at the same time with e.g. a

harvester, when the plant species have different ripening times, need different handling

methods and are used in different perspectives (Liebman and Dyck, 1993, Perez-Garcia and

del Castillo, 2016).

1.2.2 Monoculture

The monoculture growth system is a widely used method for maize production and in

Denmark maize is also produced as monoculture for several reasons. By using monoculture it

is possible to unify management, which makes crop production less complex and therefore

easier (Nielsen et al., 2015, Planteavl, 2003). The temperature hours of sun in Denmark are

not sufficient to produce sweet corn and as a result most of the maize is produced to animal as

immature maize and stored as silage (Nielsen et al., 2015). Maize seeds are sowed in rows

with 13-20 cm between seeds in the rows and 50-75 cm between rows, to a total of 100,000

seeds per ha. (Planteavl, 2003). The sowing time depends on location because maize is

sensitive to cold and maize with a slightly longer growth period is used in the south and

southeast part of Denmark. In these part of the country there exist a slightly higher

temperature and longer growth season (DMI, 2016, Planteavl, 2003). Start fertilizer is used to

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enhance germination and growth at first, and later fertilizing is provided on calculations of the

soils nutrient contents from the previous years (Planteavl, 2003). During the growth season

pests can infest the maize and there are used pesticides to reduce the impacts of the pest

(Planteavl, 2003). The harvest of the maize is done with a mower which cuts the whole plant.

Hereafter the harvested maize is laid as ensilage. When harvesting the dry substance in the

plant should preferably be around 30% (Planteavl, 2003).

1.3 Phyllophaga vetula

Phyllophaga is a genus of arthropods which contain 369 reported species (Jackson and Klein,

2006), where P. vetula is one of the species. The lifecycle of Phyllophaga spp. can have one

to several years, where P. vetula has a 1-year lifecycle (Guzman-Franco et al., 2012, Sapkota,

2006, Villalobos, 1999). The P. vetula larvae stage starts after larval hatch in June, consists of

three instars phases, before reaching pupation in late fall and thereafter maturity in spring

(Villalobos, 1999). The three instar phases indicate that the previous instar phase molt into the

following phase to continue the lifecycle and to be able to increase the body weight. The

farming practice of maize in Mexico matches that of Phyllophaga spp.’s including P. vetula’s

life cycle (Pineda et al., 2012) because of the rainy period. If the sowing and harvest time is

modified the larvae is capable of adapting and going to pupation, when the feed source stop to

exist which is normally in november (Hance et al., 1990).

The impacts by Phyllophaga spp. are severe economic losses, and is not only isolated to

maize crops but also a range of other crops (Guzman-Franco et al., 2012, Radcliffe, 1971,

Sapkota, 2006). The damage of P. vetula larvae is highly sensitive to the soil’s texture and

moisture. As these factors are not uniform throughout a field the damage is therefore not

uniform either (Katovich et al., 1998, Sapkota, 2006). The characterisation of a larvae attack

is seen by abnormal plant heights because of the root feeding of the maize plant. If the root

system is damaged the plants can be pulled from the ground with little effort, and if the root

system is badly damaged the plants can tilt themselves (Sapkota, 2006). Other

characterization such as miscolouring leaves to orange-yellow colours (Sapkota, 2006) and

final wilting of the plants because of the missing root system which inhibits water and

nutrient uptake (Sapkota, 2006).

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1.4 Arbuscular Mycorrhiza Fungi

AMF is part of Glomeromycota which can be traced back to the time, when plants started to

inhabit the earth. This happened 460 million years ago (Redecker et al. (2000) and it is

presumed that the fungus is even older. AMF exist in a mutualistic symbiosis with 80% of

the plants today (Gianinazzi et al., 2010). Among the 20% which do not form a symbiosis the

cruciferous e.g. rapeseed is to be found. The crops spinach, beetroot and beets which belong

to the amaranth family, does not form symbiosis with AMF either (Ravnskov and Larsen,

2007). AMF has mycelium in two environments; intraradical in the roots and extraradical in

the mycorrhizosphere (Smith and Smith, 2011b). The intraradical mycelium form arbuscules,

vesicles and hyphae in the plant roots, in which the arbuscules located inside the cells and

transfer nutrients between the fungi and plant species and the vesicles are used as nutrient and

metabolites storage (Smith and Smith, 2011b). The extraradical mycelium form hyphae and is

capable of searching the soil for nutrients and water (Smith and Smith, 2011b). Even though

the symbiosis enhances nutrient uptake, the plant species is still capable of nutrient uptake

through its root hair. This nutrient pathway through the root hair is called direct while the

nutrient pathway through the mycelium is called indirect. The root hair is only located close

behind the root apex whereas the colonization of AMF can happen in several places of the

roots and as such increase the depletion zone drastically (Smith and Smith, 2011b).

This mutualistic symbiosis AMF has with one host plant, it can create with several plants and

also of different species at the same time (Jakobsen, 2004, Malcova et al., 1999, Walter et al.,

1996). This mycelium network can transfer nutrients from one plant into the roots of another

plant (Jakobsen, 2004). In a way this makes AMF capable of control the nutrients uptake in

host plants but also a re-allocation which can enhance or inhibit a carbon:nutrient rate for the

plants for better or worse (Jakobsen, 2004).

1.5 Organic and inorganic fertilization of plants

Fertilization consists of nutrients which the plants need to exercise their full growth potential.

If fertilization was not given to the soil, the level of the different nutrients in the soil would

have an impact on the growth of the plants, and this often results in e.g. yield losses (Hillel,

2008). The nutrients can be divided into micro- and macro nutrients. Micronutrients are

applied with less than 1 kg/ha/year and macronutrients are applied with more than 1

Side 17 af 60

kg/ha/year. The macro nutrient includes nitrogen (N), Phosphorous (P), Potassium (K), Sulfur

(S), Calcium (Ca) and Magnesium (Mg). The micro nutrients includes iron (Fe), manganese

(Mn), boron (B), zinc (Z), copper (CU), chlorine (Cl), molybdenum (Mo), cobalt (Co) and

nickel (Ni) (Hillel, 2008).

Fertilizer can either be of organic or inorganic origin. The inorganic fertilizer is made

industrial and also called mineral fertilizer, because it does not contain carbon (C) but consists

of specific nutrients like N, P, K and S. The organic fertilizer comes from livestock and green

manure and it contains C and has a more complex nutrient composition than mineral fertilizer.

Maize also needs fertilizer, either organic or inorganic, and under Danish farming conditions

maize has a N demand around 140 kg per ha., a P demand around 25 kg per ha., a K demand

around 140 kg per ha., a Mg demand around 10 kg per ha. and a S around 10 kg per ha

(Planteavl, 2003).

1.6 Interactions

The root-feeding strategy of P. vetula can have severe negative effect on crop yield, which

can be reduced with 20-50% (Cruz-Lopez et al., 2001, Villalobos, 1999). The larvae are

present in the whole growth period of maize from seed till harvest because maize is sowed in

spring/late spring This is only around a month before the eggs hatches and the P. vetula larvae

emerges (Planteavl, 2003, Pineda et al., 2012), and when it is time for harvest of the maize

plants, it is also getting colder. These two factors influence on the larvae which will go from

the third instar phase to the pupae phase in this period (Villalobos, 1999). Even though the

larvae are present during the whole growth season, climatic parameters affect where in a field

the larvae damage the crop (Sapkota, 2006). Water is an important factor. If the water level is

low, the larvae will be lower in the soil column and disturb less than if the water level is

higher. In the last case the larvae can eat through the roots and might disconnect them at a

higher level (Sapkota, 2006).

AM symbiosis may influence the yield potential (Sabia et al., 2015), by increasing the

nutrient pool in maize which can increase the metabolisms rates in maize (Subramanian and

Charest, 1995). Most studies have investigated the ability of the AM symbiosis to increase the

uptake of P (Sabia et al., 2015, Smith and Smith, 2011a), but other studies also suggest that

Side 18 af 60

uptake of other nutrient and water is enhanced by AMF colonization (Zhao et al., 2015). This

increased nutrient uptake can come from an altered depletion zone where the maize root is not

able to reach, but the extraradical mycelium is able to reach (Smith and Smith, 2011b). This

may affect the nutrient composition of the maize plant (Azaizeh et al., 1995). However, it is

the nutrient transfer which decides the nutrient composition in maize. If maize receives fewer

nutrients from the AMF than it uses for producing the carbon molecules transferred to the

AMF, a growth depression in the maize plant can take place. This leads to less biomass

production because of the less carbon molecules available for the maize plants (Johnson et al.,

1997).

Several studies have shown that intercropping of maize with legumes increases the yield

potential over monoculture (Adesoji et al., 2013, Fawusi et al., 1982), but other studies have

also shown that intercropping maize with legumes leads to a decrease in yield (Chui and

Shibles, 1984, Gangwar and Sharma, 1994). These results are in an inconclusive conclusion,

but Postma and Lynch (2012) and Zhang et al. (2014) conclude that the complimentary root

systems and architecture structures are advantages in the milpa growth system and increase

the yield potential of maize because the plant species involved exploit the depletion zone to

the fullest without or with a minimum of competition. In Postma and Lynch (2012) it is

furthermore concluded that an increase in maize plant biomass can result in a decrease in bean

plant biomass.

Few articles report about the P. vetula and AMF interaction regarding biomass alteration in P.

vetula, but in Zitlalpopoca-Hernandez et al. (2017) who investigated the multitrophic

interactions of AMF, Entomopathogenic fungi (EPF), maize and P. vetula, AMF colonization

showed to have no impact on the biomass of P. vetula. This can be related to Currie et al.

(2011) and (Gange, 2001), who concluded that AMF colonization and biomass of pest did not

have a interaction, even though they did not investigate Phyllophaga spp. or maize. However,

it does not seem that AMF colonization can influence P. vetula growth and the presence of P.

vetula may influence the AMF colonization percentage which may indicate that the larvae

prefer eating AMF colonized roots (Zitlalpopoca-Hernandez et al., 2017).

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P. vetula is a known common pest in maize and has severe economic impacts (Cruz-Lopez et

al., 2001, Sapkota, 2006, Zitlalpopoca-Hernandez et al., 2017). However, a comparison of

how a maize monoculture growth system and a Milpa growth system or maize/legume

intercropping growth system are affected by the presence of P. vetula or Phyllophaga spp. is

not after the best of knowledge to be found in the literature.

The monoculture practices reduce the diversity of AMF species, whereas with an increase in

plant diversity increases the AMF diversity in the soil (Burrows and Pfleger, 2002). By

intercropping maize with a legume it can lead to over-yielding (Li et al., 2007) because of the

increased nutrient uptake. The AMF colonization of plant roots enhances nutrient uptake, but

also reallocate nutrients in one plant into another through the mycelium network (Walter et

al., 1996, Malcova et al., 1999), and with a complimentary roots system and structure (Postma

and Lynch, 2012) the various nutrient uptakes enhancements enhance the plant biomass. If the

intercropping system contains legumes the N-uptake increases in the system because the N

can be distributed to the non-legume plant species in the intercropping system through the

mycelium network. These results of AMF facilitating the plant species, present in an

intercropping system, are thought to be the AMF diversity rather than plant-AMF specific

species interaction (Johnson et al., 1992, Montesinos-Navarro et al., 2012), because a specific

plant-AMF species symbiosis can function as a parasitism relation (Johnson et al., 1997)

When not applying fertilizer to a field it can cause severe damage on the development of the

maize plant, and can half the yield output compared to optimal applied fertilizer (Liu et al.,

2017, Martinez et al., 2017). If fertilizer is not applied to a field the crop has to develop with

the nutrient level accessible and already in the soil. The development of a maize plant is

therefore diminished (Debreczeni et al., 2009) and the yield output and biomass production

are reduced. Studies which have investigated applied fertilizers effect on the growth of P.

vetula are after the best of knowledge not been able to be found. However, other pests are

shown to be affected by applied fertilizer (Abdallah et al., 2016, Arshad et al., 2013) where

the abundance of the Heteronychus arator larvae, from the scarabaeidae family, was

significantly affected by the type of fertilizer (Abdallah et al., 2016). If the abundance of the

larvae were affected it could be the thought that biomass growth of the larvae might also be

affected by fertilizer type.

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AMF root colonization percentage would not be affected by the type of fertilization (Cheng et

al., 2013, Dai et al., 2013, Qin et al., 2015), the soil mycelium would, however, increase with

manure as fertilizer compared to NPK fertilizer (Qin et al., 2015). This might be a response to

a more complex composition of the fertilizer. Several studies have investigated what effect

AMF can have in different P-input systems, and in Almagrabi and Abdelmoneim (2012) the

increased P-input do not negatively affect AMF colonization and beneficial abilities, while

Hagh et al. (2016) concluded that with an increase in P-input would reduce the AM symbiosis

function.

1.7 Hypothesis

The Milpa growth system will as compared to monoculture reduce the negative impact of P.

vetula on maize because of the diversity of the intercropping system. Further, the P. vetula

larvae would be influenced by a changed biotic (AMF) and abiotic (Fertilizer) soil

environment.

1.8 Objective

The objective of this study is to investigate the use of the Milpa growth system to reduce the

negative impact of P. vetula on maize as compared to the damage cause by P. vetula in maize

grown in monoculture. Moreover the influence of a change in the biotic (AMF) and abiotic

(fertilizer) soil environment on the interaction between maize and P. vetula is studied.

1.9 Research questions 1. Will maize get advantage of the Milpa growth system?

2. How will P. vetula be affected by the two maize growth systems? Milpa and

monoculture.

3. Will the maize be affected by the presence of P. vetula?

4. Will AMF effect the maize growth?

5. Will the AMF colonization be effected by maize in different growth systems and

different crops?

6. Will P. vetula reduce the AMF colonization?

7. Will AMF be able to have an impact the P. vetula growth?

8. Will fertilizer have an influence on P. vetula growth?

Side 21 af 60

2 Material and Method

2.1 Organisms

2.1.1 Plants

Three plant species were used in the experiments. In exp. 1 and 2 maize (Zea mays) was of

the variety DK-2061, which also was the same variety from which the roots feeded to the

larvae in exp. 4 originated from. Furthermore in exp. 1 and 2 there was used a bean

(Phaseolus vulgaris) of the variety 5 de Junio, and a squash (Curcubita Pepo) of the variety

Calabaza larga (Grey zucchini). In exp. 3 there was used a different variety of maize which

was Antilope.

2.1.2 Phyllophaga vetula

The P. vetula larvae were collected in Cantrabia, Zacápu, Michoacán in Mexico, the 12th

of

august 2016. This zone was chosen because of the high presence of the specie which is

estimated to 90% or more of the Phyllophaga genus. The high occurrence of P. vetula in this

area is facilitated by the landscape shape and climate conditions as the area forms a valley to

where excess water from the rainy season run down. This gives a high water level which P.

vetula is the only one of its genus that can survive in that area.

To be sure the collected larvae were not infected with entomopathogenic fungi or nematodes

they were individualized and put in quarantine in containers with humus there was watered.

During the quarantine the larvae were fed with pieces of carrot, of about 0.3 g, which were

Picture 2. Collecting of the P. vetula larvae in Cantrabia,

Zacápu, Michoacán in Mexico with the local farmer. Picture 1. Determining if the larvae were of the

P. vetula specie.

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replaced with a fresh piece every three days. The carrots were peeled before use so potential

fungicides were removed. Also, during the quarantine, the larvae were determined if they

were of the specie P. vetula based on the presence of palidia in the last abdominal segment

(Raster) and anal aperture morphology (Aragón and Morón, 2004).

2.1.3 Arbuscular mycorrhiza fungi

The AMF populations used in the experiments were exclusively the native population of the

soils, Cruco and Zacapú.

2.2 Experimental designs

2.2.1 Experiment 1

Table 1. The experimental design of exp. 1. The first column shows the Growth system, where half the pots were

monoculture maize (÷) and the other halves are Milpa growth system (+). The P. vetula column is the second

factor where half the pots had added P. vetula larvae (+) while the other halves did not have added larvae (÷).

Four treatments and six replications which add up to 24 units.

Growth system P. vetula Replications

÷ ÷ 6

÷ + 6

+ ÷ 6

+ + 6

SUM Units 24

Exp. 1 consisted of four treatments whereas two were of monoculture growth system while

the other two were of the Milpa growth system and the last factor was P. vetula larvae which

were added in two of the pots and were not added in the other two pots. Exp. 1 was a Random

Block design, with six replications. The blocks were rotated one position clockwise once per

week.

2.2.2 Experiment 2

Exp. 2 consisted of six treatments where every third pot was sowed with a maize seed while

another third of the pots were sowed with bean seeds and the last third pots were sowed with

squash seeds. P. vetula larvae were added to every second pot while the other half of the pots

were not added the larvae.

Side 23 af 60

Exp. 2 had a Random Block design, with four replications. The blocks were rotated one

position clockwise once per week.

Table 2. The experimental design of exp. 2. Crop included

of the pots as maize plants (M),

as Bean plants (B)

and the last

as squash plants (S). The P. vetula column is the second factor where half the pots had added P.

vetula larvae (+) while the other halves did not have added larvae (÷). Six treatments and four replications which

add up to 24 units.

Crop P. vetula Replications

M ÷ 4

M + 4

B ÷ 4

B + 4

S ÷ 4

S + 4

SUM Units 24

2.2.3 Experiment 3

Table 3. The experimental design of exp. 3. The first column, Phosphorous, shows half the pots got added

Phosphorous (+) and the other halves were not added phosphorous (÷). The P. vetula column is the last factor

where half the pots had added P. vetula larvae (+) while the other halves did not have added larvae (÷). Four

treatments and four replications per treatments which add up to 16 units.

Phosphorous P. vetula Replications

÷ ÷ 4

÷ + 4

+ ÷ 4

+ + 4

SUM Units 16

Exp. 3 consisted of four treatments where phosphorus was also applied to half the pots and

not to the other half. The same was about P. vetula where the larvae were added to half the

pots and not to the other half like in the two previous experiments. Exp. 3 had a Random

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Block design, with four replications. The blocks were rotated one position clockwise once per

week.

2.2.4 Experiment 4

Exp. 4 contained six treatments. There were two control treatments, where the first was not

feeded with any feed (CN), and the second was feeded with carrot (CC). In the last four

treatments one was feeded with mineral fertilized roots without AMF (MN) and another with

AMF (MW). The last two treatments were feeded with organic fertilized roots without AMF

(ON) and another with AMF (OW). The feeding of the larvae happened every three days. The

roots were cut frozen, and then thawed in water. Here after the roots were divided in seven

piles of 0.6 g, one for each replication. In treatment CN they did not receive any feed, while

the larvae in treatment CC received a piece of carrot piece on 0.3 g. However, before adding

the new feed, the old piece was removed, and checked if the larvae were still alive and active.

The experiment was a continuous of the quarantine so the larvae were kept in the same

transparent containers throughout the experimental time and kept in the same box to darken

the surroundings.

Table 4. The experimental design of exp. 4. There was one factor, feed. Within the feed factor there were seven

levels of feed types, which P. vetula larvae were feed. The levels were no feed (CN), carrot (CC), mineral

fertilized roots with none AMF (MN), mineral fertilized roots with AMF (MW), organic fertilized roots with

none AMF (ON) and organic fertilized roots with AMF (OW). Six treatments and seven replications which adds

up to 42 units.

Larvae Feed/Treatments Replications

P. vetula Nothing (CN) 7

P. vetula Carrot (CC) 7

P. vetula Mineral fertilized maize roots with none AMF (MN) 7

P. vetula Mineral fertilized maize roots with AMF (MW) 7

P. vetula Organic fertilized maize roots with none AMF (ON) 7

P. vetula Organic fertilized maize roots with AMF (OW) 7

SUM Units 42

Side 25 af 60

2.3 Experimental setup

2.3.1 Soil and sand

In exp. 1 and 2 came the soil from Cruco, Michoacán, Mexico and the sand from

Zinapecauro. In exp. 3 the soil came from Zacapú, Michoacán, Mexico and the sand came

from Zinapecauro. After the soil and sand had dried in the greenhouse on plastic covers in

exp. 1, 2 and 3, it was sieved in a 1x1 cm grid.

In exp. 4 came the soil and sand also from Cruco and Zinapecauro, as in exp. 1 and 2. To

remove the AMF from the soil, the soil and sand were sterilized in a Pro-Grow – Electric soil

sterilizer model SS-30.

2.3.2 Pots preparation

Pots were filled with a soil:sand (1:1) mixture. In exp 1, 2 and 3 had the pots had two layers

(Fig. 1A); where the bottom layer (Fig. 1A1) was soil and the top layer (Fig. 1A2) was sand.

Figure 1. This show the order of soil and sand applied to the pots in exp. 1, 2 and 3, where Pot A contains 50%

soil (A.1) and 50% sand (A.2)

Before soil and sand could be added to the pots, transparent plastic bags were put in the pots

to maintain future irrigation. Exp. 1 and 2 were similar but the sizes of the pots varied from a

3L pot with 2.5kg of substrate in exp. 1 and a 1.5L pot with 1.2kg of substrate, and in exp. 3 a

1.5L pot was filled with 1.1kg of substrate. In exp. 4 were filled the same way, except that

half the pots contained sterilized soil and sand and the other half contained non-sterilized soil

and sterilized sand. The specific amount of soil and sand added to the pots were 1250g soil

Side 26 af 60

and 1250g sand in the pots of exp. 1. In exp. 2 the pots contained 600g of soil and 600g sand.

In exp. 3 the pots contained 550g of soil and 550g sand

2.3.3 Nutrient solutions and fertilization

The solutions used in the experiments (Table 5) where only the solution I, II, III and VI were

applied, because solution IV and V containd N and P, respectively. These were excluded to

promote nodule production and AMF colonization, respectively. The applied amount was

corresponding to the supply for each solution in both exp. 1 and 2.

Table 5. The nutrients solutions used in the experiments, where both macro and micro nutrients are being used.

Each of the solutions contains chemical compounds, which are diluted in distilled water, and applied to the

substrate at different supply amounts.

Solution Chemical Concentration (g/L water) Supply (mL/kg soil)

I 61.72 6

II 25 3

III

3.5

1.8

0.7

0.06

3

IV 95.24 -

V 87.86 -

VI 135.14 3

The solutions used in exp. 1 were I, II, III and VI which were applied two days before sowing.

In exp. 2 the same solutions were applied the same day of sowing but before sowing. The

nutrients applied in exp. 3 were solution I, II, III, IV and VI. In half the pots, phosphorous

was not applied but the other half got applied 200 mg of phosphorous which is corresponding

to 10 mL of solution V. When the nutrient solutions were applied, the substrate was left to dry

before the plastic bags were taken out of the pots, shaken to mix soil and sand, and hereafter

put the bags back in the pots. The fertilization used to produce the roots used as feed in exp.

4, was organic fertilizer (Lombricomposta) in half the pots, and in the other half was used

mineral fertilizer (NPK).

Side 27 af 60

2.3.4 Sowing

After the nutrients were added and just before sowing the growth substrate was watered till

field capacity, and hereafter sowing holes were made by the end of a pen to a depth of two

cm. In exp. 1 the sowing was done by placing two seeds per desired plant where the two

similar seeds (Maize, bean and squash) were sowed next to each other in a triangular position

with the other two pair of seeds. The reason for sowing more than needed of the seeds was to

make sure that one would germinate. The same was done in exp. 2 and 3 with three seeds

instead of two per plant. In exp. 2 the triangular position was reused to position the seeds but

only one seed per position. However in exp. 3 the three seeds were sowed together in the

middle of the pot.

Dates of sowing were the 22nd

of august 2016 for exp. 1, 25th

of august 2016 for exp. 2 and 9th

of September 2016 for exp. 3.

2.4 Growth conditions

2.4.1 Climatic conditions

The climatic conditions were those which existed in Morelia, Michoacán, Mexico at the time

of the experimental period, because the experiment took place here. All the pot experiments

were placed in the greenhouse at Agroecología at the department of Investigaciones en

Ecosistemas y Sustentabilidad (IIES) at Universidad Nacional Autónoma de Mexico, Campus

Morelia (UNAM, Morelia), while the fourth small experiment was kept in the laboratory.

2.4.1.1 Day length

The day length when sowing was 12:40:18 hours and 11:28:58 hours at the day of harvest for

exp. 1. For exp. 2 it was 12:37:11 at the sowing day and 11:16:47 at the day of harvest for

exp. 2. In exp. 3, which were sowed last, the day length was reduced to 12:21:04 for the

sowing day and 11:23:08 for the harvest day (Timeanddate.com, 2017).

2.4.1.2 Temperature

The experimental period lasted from the end of august to the start of November for the pot

experiments. In these months the temperature in Morelia were 18.4°C, 18.1°C, 17.4°C and

16°C for August, September, October and November, respectively. The average temperatures

Side 28 af 60

were 10.7°C, 11.1°C, 13.4°C and 15.4°C, respectively, for the months (climatemps.com,

2017).

To increase the temperature during the night, curtains were dropped in front of the windows

in the greenhouse, which were nets. During the day the curtains were raised, to prevent

overheating the plants.

2.4.1.3 Watering

As the experiments were placed in a greenhouse there was not any precipitation. Therefore the

pots got irrigated, and the irrigating of the pots was done daily in exp. 1, 2 and 3 .The pots in

exp. 1 and 2 were watered up to 70% of the field capacity of the substrate. The field capacity

in water weight was 630g and 302g, respectively, but the pots were watered up to weight

which was done by making sure the total weight of the pots were 3190g and 1530g,

respectively. The pots weighed 60g and 28g in exp. 1 and exp. 2, respectively. While in exp.

3, the field capacity of the substrate was not know at the point of the experimental period and

therefore the watering of the pots was based on the immediate appearance of the substrate.

However, all the pots could be watered twice or thrice a day if necessary.

2.4.2 Reallocation and removing of plants

Even though more seeds were planted than necessary in some of the pots, where beans were

sowed, they did not germinate. In other pots, where both or all three bean seeds had

germinated, the excess plants were moved to other pots that needed a bean plant. The

reallocation was also done between exp. 1 and 2, however, plants where moved to pots, which

had the same treatments. The reallocation was done before adding any P. vetula larvae to the

pots. If the reallocation was not done, the bean pots in exp. 2 would have had fewer than three

replicates. The trimming of the beans happened the 16th

of September 2016 in both exp. 1 and

2, 25 and 22 days, respectively, after sowing. The trimming of both maize and squash

happened the 7th

and 12th

of September 2016 in exp. 1 and 2, respectively.

2.4.3 Adding of P. vetula

The quarantine period for the larvae used in exp. 1 lasted for 39 days for the first three larvae

and 50 days for the last two larvae. These extra larvae were added after it was discussed if the

first three larvae would have an impact on the plants in the growth systems because more than

Side 29 af 60

three larvae were collected per maize plants when collecting the larvae. The two larvae which

were added in exp. 2 had a quarantine period of 43 days, and exp. 3 had added three larvae

after a quarantine period for 50 days. In exp. 4 treatments CN, CC, MN and MW had a

quarantine period for 31 days, and treatments ON and OW had a quarantine period for 36

days. This should not affect the statistical analysis because the experiment took place in the

middle of their ecological feeding period.

2.4.4 Extra nutrients

During the experimental period, the plants in exp. 1 and 2 were added N to prevent N

deficiency. 3mL of solution IV was applied per pot

twice for exp. 1 at day 17 and 26 after sowing and once for exp. 2 at day 23 after sowing. The

deficiency was expected to happen, because N is a macronutrient and hence a very important

nutrient.

2.4.5 Unwanted pest

An unwanted pest, Spodoptera frugiperda larvae, got into the greenhouse and in order to

remove the pest a decision was made to use the pesticide Denim which contain the active

agent Benzoat Emamectin. The concentration used was 10mL/5L water and was applied by

atomizing the liquid foliar. The pest was discovered twice, the 12th

of September and the 21st

of September, and both times the pesticide was used. To prevent future attacks net covers

were made to the pot experiments.

Picture 3. Net cover for future unwanted pest attacks. Picture 4. The extent of the

unwanted pest, where the worst

attacked maize plant is shown.

Side 30 af 60

2.5 Harvest and end of experiments

The dates of harvest of experiments 1, 2 and 3 were the 27th

of October 2016, 10th

of

November 2016 and 2nd

of November 2016, respectively, which were 67 days, 78 days and 54

days, respectively, after sowing.

In exp. 1, 2 and 3 the soil was loosened slowly so it was possible to find and separate the

larvae from each pot. After the larvae were found, the rest of the soil was washed away and

sifted to recollect roots which were loosened from the root growth system after the loosening

of the soil. Hereafter the shoot and root were separated by cutting just above the highest

positioned root.

Picture 5. Maize, bean and squash plants from a Milpa growth system pot from exp. 1.

Exp. 4 ended after 28 days at the 6th

of October 2016 for treatments CC, CN, MN and MW

and the 11th

of October 2016 for treatments ON and OW, because of the five days delayed

starting days.

2.6 Analyzes

2.6.1 Biomass of plant shoot

After the separation of the shoots from the roots, the shoots were directly weighed for their

fresh weight, and thereafter put in a drying oven at 80°C at five days for exp. 1, four days for

exp. 2 and two days for exp. 3. After the drying period the shoots were weighed again for

their dry weight.

2.6.2 Biomass of plant root

After harvest the roots where put to the freezer, for later to do fresh weight. The day of the

fresh weight, the roots were thawed in tap water, and cut in small pieces. All the excess water

Side 31 af 60

was pressed out of the roots by hand, and hereafter the fresh weight was measured. Two

grams of every root was taken out to further investigation, one gram for both AMF

colonization percentage and fatty acid analysis. In some of the roots, there were not two

grams, and here the existing amount was divided in two for the two analyzes mentioned

before. The dry weight of the roots not having sufficient amount to dry were therefore

constructed by a mean of the other samples in the treatment. This was most common in beans,

and therefore the constructed dry weight of some of the bean roots were done with other bean

roots in the same treatment.

Like with the shoots, the roots was dried at 80°C in the drying oven for two days for exp. 1,

four days for exp. 2 and three days for exp. 3. After this the dry weight was measured. The

missing two grams was constructed from the water content in each root, and thereafter added

to the final weight. In all the experiments the recollected roots were added the roots of the

plant in the corresponding pot. However in exp. 1, there were three plants per pot, and

therefore the recollected roots were divided in three and added one to each of the three plants.

2.6.3 Growth of Phyllophaga vetula

Weight growth of the P. vetula larvae was done on a precision scale with three decimals for

all of the larvae in the four experiments. This was done on the day of adding the larvae to the

pots or starting the experiment. Because there was more than one larva per pot, the starting

and final weight was an average of the larvae added and re-found in each pot of the

experiments. The day the experiments ended or were harvested the larvae were weighed again

for a final weight.

The growth length of the larvae were only conducted in exp. 4, where the larvae were

measured at the starting and ending day of the experiment. This was done with a 30 cm metal

ruler upon which the larvae were stretched by hand.

2.6.4 Arbuscular mycorrhiza colonization

The roots were investigated for their percent of AMF colonization. The previous saved 1 g

roots of every plant in exp. 1, 2 and 3 was used, and in exp. 4 there was made three 2 g root

sample of each of the four root types used as feed. All these roots were prepared by clearing

and staining them by 1) adding the roots and 20mL KOH 10% solution in a 50 mL plastic

Side 32 af 60

tube with lid and 2) placed in a 90°C water bath for around 20 min. 3) After the water bath the

roots were cleansed in tap water and strained. 4) 20mL oxygenated water were added in the

tube with the roots for an hour. 5) The oxygenated water was poured from the tubes, 6) Blue

Tripano 0.05% is poured in so the roots were covered, 7) where it stayed for 15min in a water

bath at 60°C. 8) The roots were then strained, to get the rid of the excess Blue Tripano. 9)

Finally the roots were placed in scintillation vials with glycerol covering the roots.

Total AMF colonization was estimated by using a 10cm diagonally petri dish with a 1 cm

intersected grid. The petri dish was put under a stereoscope with a stained root sample and at

least 100 crossing points of the root and the grid were counted to see if the roots were

colonized or not.

2.6.5 Statistical Analysis

Statistical analyses were conducted with Statgraphics Centurion XVII software. Plant growth

parameters were compared by a two-way analysis of variance (ANOVA), with Growth

system and P. vetula in exp. 1, Crop and P. vetula in exp. 2 and Phosphorous and P. vetula in

exp. 3 as independent variables. While P. vetula growth parameters were compared with a

two-way ANOVA in exp. 1, 2, 3 and 4.

Statistical variance test was done with Levene’s variance test, and the data had homogeneity

of variance. Data including relative numbers were log-transformed before use. DW shoots in

exp. 1, exp. 3 and DW roots in exp. 3 were also log-transformed before use. Percentage

Living in exp. 3 was square root-transformed. One-way ANOVA and Multiple Range Test

with LSD were carried out for testing variance and significance between the groups,

respectively. The α-level for significance was p < 0.05. Degrees of freedom were one for

AMF in exp. 1, 2 and 4, one for Growth system in exp. 1, two for Crop in exp. 2, one for P.

vetula in exp. 1, 2 and 3, one for Phosphorous in exp. 3 and one for Fertilizer was exp. 4.

Side 33 af 60

3 Results

The results obtained came from four different experiments, and these experiments were

numbered 1 to 4. Exp. 1 investigated if the Milpa growth system could reduce the negative

impact by the root feeding P. vetula larvae and the influence of the growth systems on the

AMF colonization. Experiment 2 was made as a complimentary experiment for experiment 1.

The Milpa plants, Maize, bean and squash, were individualized to be studied how each

interact with the P. vetula larvae and influence the AMF colonization. Experiment 4 was also

a complimentary experiment for experiment 1, where the P. vetula larvae were in focus on

how they got affected by the type of feed presented to them which was roots either AMF

colonized or not and mineral or organic fertilized roots. Experiment 3 investigated another

relevant factor such as phosphorous, and if it influenced the maize-AMF-P. vetula larvae

interaction. The results will be presented in this order.

3.1 Experiment 1

3.1.1 Biomass of maize plants

Figure 2. This diagram shows the average biomass of the shoots and roots from maize, which is grown in a

monoculture growth system or Milpa growth system and are added with or without the presence of P. vetula

(Experiment 1). Small bars indicate SD of means of n=6 replicates, except in treatments with P. vetula where

there only were n=5 replicates. Mono is expressed for monoculture maize and Milpa express maize in the Milpa

Side 34 af 60

growth system. ÷ shows the absence and + shows the presence of P. vetula. The letters show significance

between the treatments, and this was done with a LSD test. Low case letters show significance between the

shoots in the treatments, and the capital letters show significance between the roots in the treatments.

Table 6. The effect of Growth system and P. vetula on average maize shoots, roots and AMF colonization in

experiment 1. n=43. *, P≤ 0.05;**, P < 0.01; ***, P≤ 0.001. The plants were either grown in a monoculture

maize growth system, three maize plants per pot, or a Milpa growth system, which consisted of a maize, bean

and squash plant per pot.

Response Factor DF F-ratio P-value

DW AVG. Maize shoots (g) Growth system 1 49.03 ***

P. vetula 1 8.97 **

Growth system x P. vetula 1 0.36 0.56

DW AVG. Maize roots (g) Growth system 1 0.20 0.66

P. vetula 1 5.97 *


AVG. Maize colonization (%) Growth system 1 19.51 ***

P. vetula 1 42.02 ***


3.1.1.1 Shoots

The biomass of maize shoots in exp. 1 was influenced by the type of growth system (Milpa or

monoculture) and by P. vetula (Table 6). The Milpa growth system produced less biomass of

maize shoots than in the monoculture growth system during the same time (Figure 2). The

average biomass of maize shoots in monoculture maize was 5.41g and 4.83g in treatments ÷P.

vetula and +P. vetula, respectively, while it in Milpa was 3.87g and 3.01g in treatments ÷P.

vetula and +P. vetula, respectively. Even though monoculture maize was not affected by P.

vetula as Milpa maize was, 22.22% reduction in Milpa, P. vetula reduced the biomass of

shoots by 0.60g in the monoculture growth system, which is a 10.78% reduction.

3.1.1.2 Roots

While the biomass of shoots was affected by both type of growth systems and by P. vetula

larvae, biomasses of the roots were only affected by larvae (Table 6 and Figure 2). In

monoculture maize there was a tendency to have a smaller root system than in the Milpa, ÷P.

Side 35 af 60

vetula treatment, and a tendency to develop a bigger root system than the Milpa, +P. vetula

treatment (Figure 2), because they were not significantly different. The Milpa ÷P. vetula and

+P. vetula treatments were 2.49g and 2.97g, respectively, which was a significant reduction

by 16.16%.

3.1.2 AMF colonization of maize roots

The average AMF colonization percentage for maize in experiment 1 was affected by the type

of growth system and by presence of P. vetula (Table 6). AMF root colonization of maize

grown in the Milpa growth system was lower than maize in the monoculture growth system

(Figure 3), like P. vetula reduced the AMF colonization percentage when present. The

colonization percentage were 29.96%, 22.54%, 25.36% and 17.51% in treatment Monoculture

Maize, ÷P. vetula, Monoculture Maize, +P. vetula, Milpa Maize, ÷P. vetula, Milpa Maize,

+P. vetula, respectively, and treatments Monoculture Maize, +P. vetula, Milpa Maize, ÷P

were not different from each other.

Figure 3. Average AMF colonization percentage of roots from maize in monoculture growth system and maize

in Milpa growth system, grown with or without the presence of P. vetula (Experiment 1). Small bars indicate SD

of means of n=6 replicates, except in treatments with P. vetula where there only were n=5 replicates. Mono

represents the monoculture growth system while Milpa represents the Milpa growth system. ÷ shows the absence

and + shows the presence of P. vetula. The letters show significance between the treatments, and this was done

with a LSD test. Low case letters show significance between the treatments.

Side 36 af 60

3.1.3 Biomass and survival of P. vetula larvae

The biomass of P. vetula did not differ between the two different growth systems (Table 7),

and the same was the results in biomass gain and survival percentage where the survival

percentage was 84.00% in both growth systems

Table 7. Biomass, biomass gain and survival percentage of P. vetula in experiment 1, when present in either

maize monoculture growth system (Monoculture) or Milpa growth system (Milpa). Mean±SD, with SD of means

of n=5 replicates. Small case letters represent significance of response between treatments.

Treatment n Biomass (g) Biomass gain (g) Survival (%)

Monoculture 5 0.76±0.03a

0.08±0.12a

84.00±26.08a

Milpa 5 0.77±0.05a

0.01±0.08a

84.00±8.94a

3.1.4 Biomass of total plants

Figure 4. The total biomass of the shoots, roots and AMF colonization in monoculture growth system and maize,

bean and squash in Milpa growth system, grown with or without the presence of P. vetula (Exp. 1). Small bars

indicate SD of means of n=6 replicates, except in treatments with P. vetula where there only were n=5 replicates.

Mono represents the monoculture growth system while Milpa represents the Milpa growth system. ÷ shows the

absence and + shows the presence of P. vetula. The letters show significance between the treatments, and this

was done with a LSD test. Low case letters show significance between the shoots in the treatments, and the

capital letters show significance between the roots in the treatments.

Side 37 af 60

Table 8. The effect of Growth system and P. vetula on shoots and roots in experiment 1. n=43. *, P≤ 0.05;**, P

< 0.01; ***, P≤ 0.001. The plants were either grown in a monoculture maize growth system, three maize plants

per pot, or a Milpa growth system, which consisted of a maize, bean and squash plant per pot.


DW shoots (g) Growth system 1 39.70 ***

P. vetula 1 7.64 *

Growth system x P.

vetula

1 0.01 0.91

DW roots (g) Growth system 1 166.10 ***

P. vetula 1 1.93 0.18

Growth system x P.

vetula

1 0.00 0.99

AMF colonization (%) Growth system 1 0.20 0.66

P. vetula 1 5.97 *

Growth system x P.

vetula

1 1.78 0.20

3.1.4.1 Shoots

Like previous with focus on maize shoot formation in exp. 1 the total biomass shoot was also

affected by type of growth system and P. vetula (Table 8). The monoculture growth system

gave again the highest amount of shoot produced over the Milpa growth system (Figure 4).

Even though P. vetula was not giving significance within either of the growth systems the

presence of the larvae reduces 10.60% and 10.34% in monoculture and Milpa, respectively.

3.1.4.2 Roots

While the total shoot formation were affected by type of growth system and P. vetula, the

biomass of the total root system was only affected by the type of growth system (Table 8) in

experiment 1, and the monoculture growth system gave the highest biomass (Figure 5), over

both Milpa growth systems. In the treatment; monoculture ÷P. vetula there was almost twice

the amount of roots, 42.65%, than in the Milpa ÷P. vetula treatment. The same happened

between the two +P. vetula treatments, 44.73%.

Side 38 af 60

3.1.5 AMF colonization of total roots

When AMF colonizes the roots of monoculture maize and Milpa in experiment 1, the highest

colonization percentage was to be found in the monoculture growth system (Table 8 and

Figure 5). The average colonization of the monoculture growth system was 29.96% and

22.54% in treatment ÷ and + of P. vetula, respectively, which was a 24.77% reduction. The

lowest colonization percentage was found in the Milpa growth systems, where +P. vetula had

the lowest with 13.46%, which was a 34.02% reduction from the Milpa ÷P. vetula treatment,

20.40% (Table 8)

It is shown that both the type of growth system and the presence of P. vetula affected the total

average AMF colonization (Table 4).

Figure 5. Total AMF colonization percentage of roots from maize in monoculture growth system and maize,

bean and squash in Milpa growth system, grown with or without the presence of P. vetula (Exp. 1). Small bars

indicate SD of means of n=6 replicates, except in treatments with P. vetula where there only were n=5 replicates.

Mono represents the monoculture growth system while Milpa represents the Milpa growth system. ÷ shows the

absence and + shows the presence of P. vetula. The letters show significance between the treatments, and this

was done with a Multi Range LSD test. Low case letters show significance between the treatments.

Side 39 af 60

3.2 Experiment 2

3.2.1 Biomass of plants

Figure 6. The biomass of shoots and roots from maize, bean and squash grown with or without the presence of P.

vetula (Experiment 2). Small bars indicate SD of means of n=4 replicates, except in bean with P. vetula where

there only were n=3 replicates. ÷ shows the absence and + shows the presence of P. vetula. The letters show

significance between the treatments, and this was done with a LSD test. Low case letters show significance

between the shoots in the treatments, and the capital letters show significance between the roots in the

treatments.

Table 9. The effect of Crop and P. vetula on shoot biomass, root biomass and AMF colonization in experiment

2. n=23. *, P≤ 0.05;**, P < 0.01; ***, P≤ 0.001.


DW shoots (g) Crop 2 15.45 ***

P. vetula 1 0.17 0.69

Crop x P. vetula 2 2.04 0.16

DW roots (g) Crop 2 120.85 ***

P. vetula 1 2.84 0.11


AMF colonization (%) Crop 2 59.63 ***

P. vetula 1 0.04 0.82

Side 40 af 60


3.2.1.1 Shoots

When dividing up the Milpa growth system in its individual plants, maize, bean and squash,

in experiment 2, the shoot biomass were affected by the type of crop (Table 9). In the maize

treatments ÷ P. vetula and +P. vetula the shoot biomass were 6.77g and 5.24g, respectively.

This was followed close by squash, which had a shoot biomass on 5.02g and 5.55g in

treatments ÷ and +P. vetula, respectively (Figure 6). Bean had the lowest shoot biomass with

2.64g and 3.05g in treatments ÷ and +P. vetula, respectively. In these results P. vetula had a

tendency to reduce maize by 22.60% in maize, while the tendency in squash was an increase

by 10.56%. In bean P. vetula did not have an effect, but in the treatment +P. vetula the

biomass amount was 15.53% higher than the ÷P. vetula treatment

Maize ÷P. vetula had the highest biomass of shoot over squash ÷P. vetula and both bean

treatments, while only have a tendency over maize +P. vetula and squash +P. vetula. Squash

+P. vetula had a higher biomass of shoot over both bean treatments, while having a tendency

to be less than maize +P. vetula and squash +P. vetula (Figure 6).

3.2.1.2 Roots

The root system in experiment 2 was also affected by the type of crop (Table 9). P. vetula

come close to give a tendency for effect, but the interaction of type of crop and P. vetula had a

tendency for effect.

The monoculture maize was affected by the presence of P. vetula (Figure 6), which was not

the case in experiment 1, by having 4.01g and 3.34g in maize treatment ÷P. vetula and +P.

vetula, respectively, and these two biomass amount was higher than any of the two treatments

in both bean and squash. P. vetula had an effect on maize, with a 16.71% reduction of the root

system. The four treatments, bean ÷ and +P. vetula, squash ÷ and +P. vetula, were not

significant from each other with 0.70g, 0.97g, 1.17g and 0.70g, respectively, but the presence

of P. vetula gave an increase in bean root for 38.57% and a reduction in squash by 40.17%.

Side 41 af 60

3.2.2 AMF colonization of maize, bean and squash roots

The AMF colonization was affected by the type of crop (Table 9) because maize had 51.29%

and 51.51% colonization in treatment ÷P. vetula and +P. vetula, respectively while bean had

colonization percentage on 30.72% and 31.00% in treatment ÷P. vetula and +P. vetula,

respectively, and squash on 27.36% and 25.74% in treatment ÷P. vetula and +P. vetula,

respectively (Figure 7). The maize treatments were not different from each other but were

different from both treatments in bean and squash, and the bean and squash treatments were

neither different.

Figure 7. AMF colonization of roots from maize, bean and squash grown with or without the presence of P.

vetula (Experiment 2). Small bars indicate SD of means of n=4 replicates, except in bean with P. vetula where

there only were n=3 replicates. ÷ shows the absence and + shows the presence of P. vetula. The letters show

significance between the treatments, and this was done with a LSD test. Low case letters show significance

between the treatments.


The biomass of P. vetula larvae in experiment 2 were not affected by the specific plants of

maize, bean or squash (Table 10). When looking at biomass gain and survival percentage,

there was not an effect of the different plants. The survival percentage was 25%, 25% and

30% in maize, bean and squash, respectively.

Side 42 af 60

Table 10. Biomass, biomass gain and survival percentage of P. vetula in experiment 2, when present in either

maize, bean or squash. Mean±SD, with Standard Error of means of n=4 replicates, except under biomass and

biomass gain in maize and bean where only three replicates had surviving P. vetula larvae. Small case letters

represent significance between treatments.


Maize 3/3/4 0.79±0.09a

0.04±0.07a

25.00±19.15a

Bean 3/3/4 0.85±0.08a

0.11±0.14a

25.0±19.15a

Squash 4/4/4 0.90±0.03a

0.06±0.03a

30.00±11.55a

3.3 Experiment 4

3.3.1 Biomass of P. vetula larvae

Both AMF and Fertilizer had no significant effect on the end weight or the final length of P.

vetula. AMF did not have any effect on weight gain or length gain. However, fertilizer did

show an effect on weight gain and length gain (Table 12). P. vetula feeding on organic

fertilized roots increases both the weight and length gain (Table 11). All of the larvae lost

weights during the 28 days, except CC, and in treatment MRN the larvae lost more weight

than in ON. Of the treatments only MN had a negative length gain and treatments ON and

OW had a significantly increased alteration of length than MN

Table 11. Biomass, biomass gain, length and length gain of P. vetula in experiment 4. Mean±SD, with Standard

Error of means of n=7 replicates except in treatments CC and OW where only six replicates of P. vetula larvae

survived. Small case letters represent significance between treatments. The treatments were control without feed

(CN), control feeded with carrot (CC), mineral fertilized maize roots without AMF (MN), mineral fertilized

maize roots with AMF (MW), organic fertilized maize roots without AMF (ON) and organic fertilized maize

roots with AMF (OW).

Treatment n Biomass (g) Biomass gain Length (mm) Length gain (mm)

CN 7 0.79±0.09a

-0.03±0.05ab

28.43±1.27a

-0.43±1.81a

CC 6 0.84±0.12a

0.01±0.05b

30.50±1.38b

1.33±1.51bc

MN 7 0.81±0.10a

-0.07±0.03a

29.29±1.38ab

-0.43±1.40a

MW 7 0.78±0.10a -0.04±0.04

ab 28.86±1.86

ab 0.57±1.27

ab

ON 7 0.81±0.07a -0.02±0.05

b 30.14±1.46

b 2.29±0.95

c

Side 43 af 60

OW 6 0.81±0.12a -0.02±0.04

ab 29.83±1.60

ab 1.83±1.60

bc

Table 12. The effect of AMF and fertilizer on the biomass, biomass gain length and length gain P. vetula in

experiment 4. n=27. *, P≤ 0.05;**, P < 0.01; ***, P≤ 0.001. The controls CC and CN are excluded from this

statistically table.


Biomass (g) AMF 1 0.14 0.71

Fertilizer 1 0.12 0.74

AMF x Fertilizer 1 0.12 0.73

Biomass gain (g) AMF 1 1.02 0.32

Fertilizer 1 4.19 *


Length (mm) AMF 1 0.36 0.55

Fertilizer 1 2.24 0.15


Length gain (mm) AMF 1 0.29 0.59

Fertilizer 1 15.39 ***



The AMF colonization was non-existing in MN and ON, which were not supposed to have

colonization. In MW and OW there were colonization but the two treatments were not

significantly different (Table 8).

Table 13. AMF colonization percentage in maize roots used as feed in experiment 4. Mean±SD, with Standard

Error of means of n=3 replicates. Small case letters represent significance between treatments. The roots have

been fertilized with either mineral (M) or organic fertilizer (O) and either colonized (W) or not (N) by AMF. The

CN and CC treatments are controls (C) feed with none (N) or carrot (C).

Treatment Count. % Colonization

CN - -

CC - -

MN 3 0.00±0.00a

Side 44 af 60

MW 3 35.40±3.72a

ON 3 0.00±0.00

OW 3 23.30±7.91a

3.4 Experiment 3

3.4.1 Biomass of plants

Table 14. Biomass of shoots and roots and AMF colonization percentage in experiment 3. Mean±SD, with

Standard Error of means of n=4 replicates. Small case letters represent significance between treatments. The

treatments with maize had either applied (+) phosphorous or not (÷), and P. vetula was also added (+) or not

(÷).Each pot was added three P. vetula larvae.

Treatment n DW shoots (g) DW roots (g) AMF Colonization (%)

÷Phosphorous, ÷P. vetula 4/4/4 1.43±0.30a

0.89±0.24a

31.24±3.43d

÷Phosphorous, +P. vetula 4/4/4 1.39±0.10a

0.71±0.07a

20.92±1.86c

+Phosphorous. ÷P. vetula 4/4/4 7.83±1.64b

2.54±0.44b

10.89±0.85b

+Phosphorous, +P. vetula 4/4/4 6.42±0.90b

2.40±0.53b

8.46±0.44a

Table 15. The effect of phosphorous and P. vetula on the biomass of shoots and roots and AMF colonization

percentage in experiment 3. n=32. *, P≤ 0.05;**, P < 0.01; ***, P≤ 0.001. The maize plants were grown

individual in 1.1kg substrate. Each pot was added three P. vetula larvae.


DW shoots (g) Phosphorous 1 354.78 ***

P. vetula 1 1.49 0.25

Phosphorous x P. vetula 1 0.98 0.34

DW roots (g) Phosphorous 1 112.84 ***

P. vetula 1 1.58 0.23

Phosphorous x P. vetula 1 0.38 0.55

AMF Colonization (%) Phosphorous 1 515.06 ***

P. vetula 1 57.16 ***

Phosphor x P. vetula 1 2.91 0.11

Side 45 af 60

3.4.1.1 Shoots

The shoots in experiment 3 were influenced by the applied phosphorous (Table 15). The

applied phosphorous gave greater shoots production (Table 14) and P. vetula did not

influence the shoot production.

3.4.1.2 Roots

The roots in experiment 3 were affected phosphorus (Table 15) and all +Phosphorous

treatments had the greatest root production (Table 14).


In experiment 3 the presence of P. vetula as a factor gave a reduction in the colonization

percentage in maize (Table 14 and 15). Moreover, did the applied phosphorous influence the

AMF colonization percentage.


In experiment 3 there was an effect by applied phosphorous on the biomass of P. vetula where

biggest larvae were to be found in the high-P soil. In biomass gain did phosphorous not show

an effect even though the mean of the larvae in low-P soil was higher than the high-P soil

(Table 16). Survival percentage was not affected by the two phosphorus treatments (Table

16).

Table 16. Biomass, biomass gain and survival percentage of P. vetula in experiment 3. Mean±SD, with SD of

means of n=4 replicates except under biomass and biomass gain in treatments ÷Phosphorous and +Phosphorous

where only three replicates had surviving P. vetula larvae. Small case letters represent significance between

treatments. The treatments with maize were either applied (+) phosphorous or not (÷). In each pot there were

added three P. vetula larvae.


÷Phosphorous 3/3/4 1.04±0.12b

0.18±0.13a

41.67±41.94a

+Phosphorous 4/4/4 0.74±0.10a

-0.01±0.07a

100.00±0.00a

Side 46 af 60

4 Discussion

The hypothesis of this study was that the Milpa growth system in contrast to monoculture

would reduce the negative impact by P. vetula on maize plants because of the increased plant

diversity. However, the negative impact by P. vetula on maize was in present study expressed

in the Milpa growth system and not in the monoculture growth system. No study was to be

found focusing on the interaction of P. vetula or Phyllophaga spp. and Milpa or maize in an

intercropping system. Even so it was believed that the Milpa growth system could enhance

the growth of maize to withstand the biomass reduction at the presence of P. vetula because

of increased nutrient uptake (Postma and Lynch, 2012, Zhang et al., 2014). In Risch (1980) it

was concluded that the Milpa growth system resulted in a beetle abundance decrease and

Trenbath (1993) also came to the conclusion that intercropping can enhance the growth of

maize under pest pressure. This, however, is regarding foliar-feeding pests which are not

comparable to root-feeding pests. Maize, bean and squash did not influence the biomass or

biomass gain of P. vetula; however, maize had a tendency for a biomass reduction at the

presence of P. vetula while bean and squash were not affected. Could this mean that the

larvae did not just stuff them with feed but in fact picked and chose the feed and thereby

avoid using unnecessary energy (Tsunoda et al., 2017) because the plant species were

differently affected while the P. vetula larvae were not affected. It can be said that the Milpa

growth system does not reduce the pressure or influence the biomass of P. vetula on maize

but on the contrary increases the negative impacts by the P. vetula larvae on the Milpa maize.

Picture 6. The investigated interactions in the present study

Side 47 af 60

4.1 Growth systems’ effect on maize

The Milpa growth system did not increase the shoot biomass of maize as compared to the

monoculture growth system. However, the shoot biomass was lower in the Milpa growth

system compared to the monoculture growth system while the root biomass was not affected.

The total biomass was also negatively affected by the Milpa growth system but this can also

be a result of the lower biomass production in bean and squash. The lower biomass

production in bean and squash does not decrease the total biomass further when comparing

with the biomass of the maize plant in the Milpa growth system and the total biomass in the

Milpa growth system in relation to the monoculture growth system. The results in the present

study are in contrast to findings in Postma and Lynch (2012) and Zhang et al. (2014) who

concluded that the Milpa growth system would increase the biomass production in both

maize, bean and squash when compared to the monoculture growth system for each of the

plant species. The contradiction between this study and the findings in Postma and Lynch

(2012) and Zhang et al. (2014) could be due to the experimental designs. The soil volumes

differ significantly between this study and both of Postma and Lynch (2012) and Zhang et al.

(2014) which dealt with a 60cm x 60cm x 150cm pot for three plants and a field experiment,

respectively. In these greater soil volumes the complimentary root systems could unfold more

than in a 3L pot wherein the competition between the plant species could increase and be a

downfall for the Milpa growth system in this study. Further studies should be conducted to

investigate the potential biological control of the Milpa growth system towards P. vetula.

However, it is interesting to see that bean and squash did not decrease the total biomass

production any further even though it was shown that the two plant species had a lower

biomass than maize when being a part of a monoculture growth system. It can indicated that

bean and/or squash increased the biomass production in the milpa growth system compared to

monoculture and this corresponds with the findings in Postma and Lynch (2012) and Zhang et

al. (2014). To fully understand the beneficial abilities of the Milpa growth system it must be

investigated under circumstances’ where the growth system has the possibilities to unfold the

elements which could make it a beneficial growth system.

4.2 Maize plant herbivory by P. vetula

The presence of P. vetula reduced the maize shoot and root biomass in the Milpa growth

system while at the same time the presence of P. vetula both did not show and showed a

Side 48 af 60

tendency for reduction in monoculture growth system. Furthermore, the total shoot biomass of

the Milpa and monoculture growth systems was affected by P. vetula; even though it seemed

that P. vetula had no effect within either of the growth systems. The total root biomass was

not affected by P. vetula. In Villalobos (1999) there was observed a yield reduction in maize

at approximately 50%. In the present study there was measured biomass production in the

Milpa growth system which was reduced by P. vetula, but not a 50% reduction, however, it is

not known how much the reduced biomass would have influenced the yield of the maize

plants. It was previously discussed that the Milpa growth system was not able to increase the

biomass of maize or reduce the negative impacts by P. vetula on maize. P. vetula was not able

to reduce the total biomass of the Milpa growth system, even though maize and bean are host

plants to P. vetula (Sapkota, 2006). This could be associated with the tendency for biomass

increase in the squash plant, at the presence of P. vetula because the squash plant is not listed

as a host plant (Postma and Lynch, 2012, Sapkota, 2006). The missing biomass reduction by

P. vetula in monoculture maize is in contrast to other findings (Zitlalpopoca-Hernandez et al.,

2017). In Zitlalpopoca-Hernandez et al. (2017) the P. vetula larvae were added earlier in the

growth period than in the present study, and this could enhance the pressure by P. vetula.

Moreover, there were added two P. vetula larvae per plant in Zitlalpopoca-Hernandez et al.

(2017) and five larvae per three plants in this study which furthermore can enhance the

pressure on maize plants in Zitlalpopoca-Hernandez et al. (2017). The less impact inflicted by

P. vetula on the roots in relation to the shoots might be attributed to a reallocation of the

energy to the root systems for maintaining the nutrient uptake. These nutrients are used in the

plants metabolisms processes (Gray and Steffey, 1998, Subramanian and Charest, 1995)

where the Milpa maize plant cannot reallocate sufficient to maintain the root biomass. P.

vetula larvae have a greater herbivory effect on maize in the Milpa growth system than in a

monoculture growth system and this can be due to the nutrient competition between the plant

species and therefore maize cannot handle further pressure from the P. vetula. The total

biomass of the Milpa growth system is not affected by P. vetula which can be attributed to the

squash plant which showed a tendency for increasing the biomass under pressure by P. vetula.

4.3 AMF colonization in roots of Milpa or monoculture growth systems

AMF colonization in maize roots was influenced by the growth system. The AMF

colonization percent was lower in Milpa maize than in monoculture maize. The total AMF

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colonization percent in the Milpa growth system was lower than the total AMF colonization

percent in the monoculture growth system and even lower than AMF colonization percent in

the Milpa maize. Bean and squash had a lower AMF colonization percent than maize. From

the results of the present study it is not possible to say that AM symbiosis enhances a growth

depression or growth increase in maize (Jakobsen, 2004). Meng et al. (2015) showed when

intercropping maize and soybean did AMF slightly enhance biomass growth of maize but did

not enhance biomass growth of soybean. They used a 3kg substrate which was divided with

three levels of barrier – solid barrier, mesh barrier and no barrier – to investigate the effects of

inoculated AMF. In Meng et al. (2015) the AMF colonization percentage did not vary in

maize roots between monoculture and intercropping which it did in soybean roots. Even

though the AMF colonization percentage did not differ the biomass in maize increased in the

intercropping system, and the lower AMF colonization percentage in soybean roots did not

affect the biomass in the soybean plant. This is in contrast to the present study in which both

the AMF colonization percentages of maize roots and biomass of maize were negatively

affected by the Milpa growth system. A difference between this study and Meng et al. (2015)

is the squash plant. The squash plant in this study had almost the same biomass level as maize

while at the same time it had a much lower AMF colonization percentage than the maize roots

(Rouphael et al., 2015). Functional compatibility could be the reason why there is not a direct

interaction of AMF colonization percentage and biomass growth of the plant species (Boucher

et al., 1999, Sousa et al., 2012). How exactly the AMF-growth system interaction works

cannot be discussed to the fullest, e.g. if the AMF mycelium network transport of nutrient

influence the growth of the plants species in either the Milpa or monoculture growth systems

(Jakobsen, 2004, Malcova et al., 1999, Walter et al., 1996) because AMF is missing as a

factor. It can be said that AMF colonization decreased in maize roots when it is part of the

Milpa growth system, and at the same time the biomass of maize decreased. However, it

cannot be said if this is an interaction. Further studies would have to clarify if AMF enhances

growth or growth depression as the result in this study shows the Milpa growth system

decreased the maize biomass.

4.4 Herbivory by P. vetula on AMF colonized maize roots

The presence of P. vetula decreased the AMF colonization percent in the maize roots in both

the Milpa and monoculture growth system. However, this study also reported that the

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presence of P. vetula larvae did not have an effect on AMF colonization percentages in either

maize, bean or squash monoculture which therefore is in contrast to the other results

presented in this study. In the literature (Zitlalpopoca-Hernandez et al., 2017) it was reported

that the presence of P. vetula reduced the AMF colonization percentages and as such is in

agreement with the results in this study. The contrasting herbivory effect by P. vetula larvae

on the AMF colonization percent results in the present study might, however, be due to

unskilled counting abilities of the crossing points in the intersected petridish because the

results, showing no effect on the AMF colonization percentage by P. vetula in the

individualized maize, bean and squash experiment, were the first samples which were

counted. In these there were counted 500-1000 crossing points per sample which must have

been done unskilfully. This is thought because the similar experimental design of the

Milpa/monoculture growth system experiment showed a reduction in the AMF colonization

percentage, while also the third experiment with the soil type in difference showed a

reduction at the presence of P. vetula. The decreased AMF colonization percentages can be

because of increased interest by P. vetula on the colonization points roots because they

contain specific nutrients (Liu et al., 2016). If the roots are increased in content of specific

nutrients, the transfer point, the AMF colonization points, might have further increased

nutrient contents, and a following increased interest for the P. vetula larvae (Tsunoda et al.,

2017). This can be hold against the fact that the root biomass are not as much affected by the

presence of P. vetula as the AMF colonization percentages. The presence of P. vetula

decreases the AMF colonization percentages in maize roots, and this might be caused by an

increased interest in the AMF colonization points by P. vetula because the points are the place

for nutrient transfer between AMF and maize, and therefore the nutrient content are increased

for specific nutrients.

4.5 AMF and fertilizer impacts on P. vetula

4.5.1 Biomass

The feed treatments, no feed, mineral fertilized roots with and without AMF, organic

fertilized roots with and without AMF, had a negative influence on the weight of P. vetula,

while the carrot treatment, which was a control, did not have negative effect on the weight of

P. vetula. However, the carrot treatment was also expected to gain the most weight, due to the

high energy content of carrots. In the mineral fertilized roots without AMF treatment the

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larvae were losing the most weight, as opposed to organic fertilized roots with and without

AMF treatments loosed the least weight. This was related to the fertilizer type. In Stevens and

Jones (2006) they found that biomass was affected by fertilized and non-fertilized which can

go along the two fertilizer levels and the two levels of soil phosphorous presented in this

study. The nutrient composition in a plant will alter according to the nutrient availability in

the soil (Aliyu, 2000, Kadar et al., 2000, Martinez-Alcantara et al., 2016). If the nutrient

composition does not increase as much when exposed to mineral fertilizer than to organic

fertilizer, the plant produces more cellulose in the root. This is very hard to digest by any

monogastric animals, if possible at all. At the same time AMF colonized roots did not have a

significant effect on the biomass of the P. vetula (Zitlalpopoca-Hernandez et al., 2017) which

were thought to alter the nutrient composition by increasing the nutrient uptake. There have

not been made any nutrient composition analyses of the roots in this study to determine

whether AMF have made an alteration in the nutrient composition. If this was the case the

alteration was not in such a degree to have had an impact on the biomass of P. vetula in way

that the fertilizer type did.

4.5.2 Length

In the length gain results of P. vetula, mineral fertilized roots without AMF did have a

negative length as the only one, except the no feed treatment. Organic fertilized roots with

and without AMF had the greatest length gain, even greater than the carrot treatment. The

fertilizer type was again the significant factor while AMF did not have an effect. The final

length was not affected by either fertilizer type or AMF colonization. Even though Stevens

and Jones (2006) found results for an effect of fertilizer management on biomass it would be

possible to argue that fertilizer could also inflict changes in the other growth parameters of P.

vetula such as length because fertilizer type can influence on the abundance of white grubs

(Stevens et al., 2007) which means that fertilizer management can affect P. vetula. The fact

that organic fertilizer increases the length (Present study) and decreases the abundance

(Stevens et al., 2007) of white grubs, organic fertilizer might have made an alteration in the

nutrient composition of the soil and plant can change the growth of root-feeding pests

(Johnson et al., 2009, Stevens and Jones, 2006).

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5 Conclusion

In this study it was hypothesized that the Milpa growth system could decrease the negative

impact in maize by P. vetula compared to monoculture maize, and in cooperation with AMF

would the negative impacts by P. vetula decrease even more. The results showed that the

Milpa growth system could not decrease the negative impacts by P. vetula on maize.

However, the negative impacts increased when maize was part of the Milpa growth system

compared to the monoculture growth system. The reduction in Milpa maize, which was in

contrast to the literature (Postma and Lynch, 2012), is due to the lesser soil volume in present

study. The complementary root systems of maize, bean and squash could not unfold properly,

and a competition for soil nutrients must have been present. The greater herbivory of Milpa

maize by P. vetula is due to further pressure on the maize because the Milpa growth system is

already pressuring the maize plant. The total biomass is not affected by P. vetula due to a

tendency in biomass increase in squash plants at the presence of P. vetula. The AMF

colonization percentage is declining in Milpa maize compared to monoculture maize but this

does not report if the AMF are enhancing growth or growth depression in the Milpa growth

system because the growth of Milpa maize is already affected by the Milpa growth system.

The reduction of AMF colonization percentage at the presence of P. vetula larvae must be

caused by an increased interest of the P. vetula because the colonization points have a

different nutrient composition than the rest of the root system. The growth parameters of P.

vetula were not affected neither by the Milpa nor the monoculture growth system. The

presence of AMF could not affect the biomass of P. vetula either, however, the biomass gain

and length gain of P. vetula were affected by the different fertilizer type, in which the organic

fertilized roots made the P. vetula larvae lose the lowest amount of biomass and increased the

length of the P. vetula larvae.

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6 Perspectives

Even though the results of the present study showed that the Milpa growth system could not

reduce the negative impacts by P. vetula on maize, further investigations must be conducted

to fully understand if the beneficial effects of the Milpa growth system in fact could be used

as a biological control factor because results from the pot experiments are not directly

comparable with field experiments, and the results in the present study is in contrast to the

literature. Especially when the roots are the beneficial factor in the plant growth, they need

soil enough in order not to reach the defined barriers, and therefore develop as in natural

circumstances. When the roots are developing in sufficient soil, the AMF factor would also be

highlighted if they increase the plant growth of maize and thereby increase the ability of the

maize plant to withstand the feeding pressure by P. vetula.

It is possible to say that the Danish agriculture production system is industrialized, and in this

production huge machines are used due to a uniform production method. Practically it makes

it hard to implement the Milpa growth system as it contains different plant species with

different uses (Isabel Moreno-Calles et al., 2012, Ortiz-Timoteo et al., 2014), which is

problematic in a uniform production method. Harvesting three different plants would require

a harvest method for all the three plants at one time and this would be difficult. The cob, the

bean seeds, and the squash fruit all have different positions on the plants and should be treated

accordingly. This seeks a harvesting method to harvest all three objects at the same time,

treating the objects individually and storage the objects differently while keep harvesting. The

harvesting issue is not the only problem because the plant species ripen in different time

scales. To overcome this problem the plants could be sown according to growth period to

reach a simultaneous ripening time. However, sowing multiple times could influence the

already established plants.

Instead of using the specific Milpa growth system, a modified version could be more practical

to implement in the organic Danish agricultural production. E.g. a maize-bean intercropping

system could fulfil the lack of protein source in the organic cattle/dairy production because

the bean plant would increase the total N of the biomass in the field. By harvesting the two

plant species together and lay as ensilage it could later be used as increased N-feed for the

cattle. Compared to the conventional cattle/dairy production in which it is possible to import

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the protein source as soybean cakes from South America the organic cattle/dairy production

cannot use the soybeans cakes, because in the soybean production there are used pesticides.

Introducing the specific Milpa growth system to Denmark would be practical hard, but a more

general overlook for intercropping systems which could be implemented in the Danish

agricultural production could be of high interest, because it can supply a more diversified

nutrient composition. Furthermore, the increase in crop diversity increases the niches and

thereby increases associated organisms, which can perform plant beneficial effects, such as

nutrient increase, natural enemies effect towards pests etc.

Side 55 af 60

7 Bibliography

ABDALLAH, M., MWATAWALA, M. W. & KUDRA, A. B. 2016. Abundance and

dispersal of Heteronychus arator (Coleoptera: Scarabaeidae) in maize fields under

different fertilizer treatments. Springerplus, 5.

ADESOJI, A. G., ABUBAKAR, I. U., TANIMU, B. & LABE, D. A. 2013. Influence of

Incorporated Short Duration Legume Fallow and Nitrogen on Maize (Zea mays L.)

Growth and Development in Northern Guinea Savanna of Nigeria. American-Eurasian

Journal of Agricultural & Environmental Sciences, 13, 10.

ALIYU, L. 2000. Effect of organic and mineral fertilizers on growth, yield and composition

of pepper (Capsicum annuum L.). Biological Agriculture & Horticulture, 18, 29-36.

ALMAGRABI, O. A. & ABDELMONEIM, T. S. 2012. Using of Arbuscular Mycorrhizal

Fungi to Reduce the Deficiency Effect of Phosphorous Fertilization on Maize Plants

(Zea mays L.). Life Science Journal-Acta Zhengzhou University Overseas Edition, 9,

1648-1654.

ARAGÓN, A. & MORÓN, M. Á. 2004. DESCRIPCIÓN DE LAS LARVAS DE TRES

ESPECIES DE PHYLLOPHAGA (COLEOPTERA: MELOLONTHIDAE:

MELOLONTHINAE) DEL VALLE DE PUEBLA, MÉXICO. Folia Entomologica

Mexicana, 43.

ARSHAD, M. J., FREED, S., AKBAR, S., AKMAL, M. & GUL, H. T. 2013. Nitrogen

Fertilizer Application in Maize and Its Impact on the Development of Chilo partellus

(Lepidoptera: Pyralidae). Pakistan Journal of Zoology, 45, 141-147.

AZAIZEH, H. A., MARSCHNER, H., ROMHELD, V. & WITTENMAYER, L. 1995.

EFFECTS OF A VESICULAR-ARBUSCULAR MYCORRHIZAL FUNGUS AND

OTHER SOIL-MICROORGANISMS ON GROWTH, MINERAL NUTRIENT

ACQUISITION AND ROOT EXUDATION OF SOIL-GROWN MAIZE PLANTS.

Mycorrhiza, 5, 321-327.

BENNETT, A. E., ALERS-GARCIA, J. & BEVER, J. D. 2006. Three-way interactions

among mutualistic mycorrhizal fungi, plants, and plant enemies: Hypotheses and

synthesis. American Naturalist, 167, 141-152.

BOUCHER, A., DALPE, Y. & CHAREST, C. 1999. Effect of arbuscular mycorrhizal

colonization of four species of Glomus on physiological responses of maize. Journal

of Plant Nutrition, 22, 783-797.

BURROWS, R. L. & PFLEGER, F. L. 2002. Arbuscular mycorrhizal fungi respond to

increasing plant diversity. Canadian Journal of Botany-Revue Canadienne De

Botanique, 80, 120-130.

CHENG, Y., ISHIMOTO, K., KURIYAMA, Y., OSAKI, M. & EZAWA, T. 2013. Ninety-

year-, but not single, application of phosphorus fertilizer has a major impact on

arbuscular mycorrhizal fungal communities. Plant and Soil, 365, 397-407.

CHUI, J. A. N. & SHIBLES, R. 1984. INFLUENCE OF SPATIAL ARRANGEMENTS OF

MAIZE ON PERFORMANCE OF AN ASSOCIATED SOYBEAN INTERCROP.

Field Crops Research, 8, 187-198.

CLIMATEMPS.COM. 2017. http://www.morelia.climatemps.com/temperatures.php [Online].

[Accessed 31-03 2017].

CRUZ-LOPEZ, J. A., CASTRO RAMIREZ, A. E., RAMIREZ SALINAS, C. & GOMEZ Y

GOMEZ, B. 2001. Manual suppression of adults of Phyllophaga spp. and Anomala

spp. on maize in Mexico. Manejo Integrado de Plagas, 41-47.

http://www.morelia.climatemps.com/temperatures.php

Side 56 af 60

CURRIE, A. F., MURRAY, P. J. & GANGE, A. C. 2011. Is a specialist root-feeding insect

affected by arbuscular mycorrhizal fungi? Applied Soil Ecology, 47, 77-83.

DAI, J., HU, J. L., LIN, X. G., YANG, A. N., WANG, R., ZHANG, J. B. & WONG, M. H.

2013. Arbuscular mycorrhizal fungal diversity, external mycelium length, and

glomalin-related soil protein content in response to long-term fertilizer management.

Journal of Soils and Sediments, 13, 1-11.

DEBRECZENI, K., BERECZ, K. & MIHALOVICS, M. 2009. EFFECT OF NUTRIENT

DEFICIENCY AND SURPLUS ON WHEAT AND MAIZE PRODUCTION. Cereal

Research Communications, 37, 85-88.

DMI, D. M. I. 2016. http://www.dmi.dk/vejr/arkiver/vejrarkiv/ [Online]. [Accessed 13-04

2017].

DWIVEDI, A., SINGH, A., NARESH, R. K., KUMAR, M., KUMAR, V., BANKOTI, P.,

SHARMA, D. K., THANESHWAR, SINGH, A. & SINGH, O. 2016. Towards

Sustainable Intensification of Maize (Zea mays L.) plus Legume Intercropping

Systems; Experiences; Challenges and Opportunities in India; A Critical Review.

Journal of Pure and Applied Microbiology, 10, 725-740.

EKESI, S., MANIANIA, N. K., AMPONG-NYARKO, K. & ONU, I. 1999. Effect of

intercropping cowpea with maize on the performance of Metarhizium anisopliae

against Megalurothrips sjostedti (Thysanoptera : Thripidae) and predators.

Environmental Entomology, 28, 1154-1161.

FAOSTAT. 2014. CROPS [Online]. http://www.fao.org/faostat/en/#data/QC. [Accessed 11-

04 22017].

FAOSTAT 2015. Crops. Fao Statistical Pocketbook.

FAWUSI, M. O. A., WANKI, S. B. C. & NANGJU, D. 1982. PLANT-DENSITY EFFECTS

ON GROWTH, YIELD, LEAF-AREA INDEX AND LIGHT TRANSMISSION ON

INTERCROPPED MAIZE AND VIGNA-UNGUICULATA (L) WALP IN

NIGERIA. Journal of Agricultural Science, 99, 19-23.

GANGE, A. C. 2001. Species-specific responses of a root- and shoot-feeding insect to

arbuscular mycorrhizal colonization of its host plant. New Phytologist, 150, 611-618.

GANGWAR, K. S. & SHARMA, S. K. 1994. FODDER-LEGUME INTERCROPPING IN

MAIZE (ZEA-MAYS) AND ITS EFFECT ON SUCCEEDING WHEAT

(TRITICUM-AESTIVUM). Indian Journal of Agricultural Sciences, 64, 38-40.

GIANINAZZI, S., GOLLOTTE, A., BINET, M.-N., VAN TUINEN, D., REDECKER, D. &

WIPF, D. 2010. Agroecology: the key role of arbuscular mycorrhizas in ecosystem

services. Mycorrhiza, 20, 519-530.

GOMEZ-ARROYO, S., MARTINEZ-VALENZUELA, C., VILLALOBOS-PIETRINI, R. &

WALISZEWSK, S. 2011. Pesticides: genotoxic risk of occupational exposure: en:

pesticides: the impacts of

pesticide exposure, http://www.intechopen.com/books/pesticides-the-impacts-of-

pesticides-exposure/pesticides-genotoxic-risk-ofoccupational-exposure, Intech.

GRAY, M. E. & STEFFEY, K. L. 1998. Corn rootworm (Coleoptera : Chrysomelidae) larval

injury and root compensation of 12 maize hybrids: an assessment of the economic

injury index. Journal of Economic Entomology, 91, 723-740.

GUZMAN-FRANCO, A. W., HERNANDEZ-LOPEZ, J., ENRIQUEZ-VARA, J. N.,

ALATORRE-ROSAS, R., TAMAYO-MEJIA, F. & ORTEGA-ARENAS, L. D. 2012.

Susceptibility of Phyllophaga polyphylla and Anomala cincta larvae to Beauveria

bassiana and Metarhizium anisopliae isolates, and the interaction with soil properties.

Biocontrol, 57, 553-563.

http://www.dmi.dk/vejr/arkiver/vejrarkiv/

http://www.fao.org/faostat/en/#data/QC

http://www.intechopen.com/books/pesticides-the-impacts-of-pesticides-exposure/pesticides-genotoxic-risk-ofoccupational-exposure

http://www.intechopen.com/books/pesticides-the-impacts-of-pesticides-exposure/pesticides-genotoxic-risk-ofoccupational-exposure

Side 57 af 60

HAGH, E. D., MIRSHEKARI, B., ARDAKANI, M. R., FARAHVASH, F. & REJALI, F.

2016. Optimizing phosphorus use in sustainable maize cropping via mycorrhizal

inoculation. Journal of Plant Nutrition, 39, 1348-1356.

HANCE, T., GREGOIREWIBO, C. & LEBRUN, P. 1990. AGRICULTURE AND

GROUND-BEETLES POPULATIONS - THE CONSEQUENCE OF CROP TYPES

AND SURROUNDING HABITATS ON ACTIVITY AND SPECIES

COMPOSITION. Pedobiologia, 34, 337-346.

HILLEL, D. 2008. Soil Fertility and Nutrition. Soil in the Environment - Crucible of

Terrestrial Life. Academic Press.

HUND, A., RUTA, N. & LIEDGENS, M. 2009. Rooting depth and water use efficiency of

tropical maize inbred lines, differing in drought tolerance. Plant and Soil, 318, 311-

325.

ISABEL MORENO-CALLES, A., CASAS, A., GARCIA-FRAPOLLI, E. & TORRES-

GARCIA, I. 2012. Traditional agroforestry systems of multi-crop "milpa" and

"chichipera" cactus forest in the arid Tehuacan Valley, Mexico: their management and

role in people's subsistence. Agroforestry Systems, 84, 207-226.

JACKSON, T. A. & KLEIN, M. G. 2006. Scarabs as pests: A continuing problem.

Coleopterists Bulletin, 60, 102-119.

JAKOBSEN, I. 2004. Hyphal fusion to plant species connections - giant mycelia and

community nutrient flow. New Phytologist, 164, 4-7.

JOHNSON, N. C., COPELAND, P. J., CROOKSTON, R. K. & PFLEGER, F. L. 1992.

MYCORRHIZAE - POSSIBLE EXPLANATION FOR YIELD DECLINE WITH

CONTINUOUS CORN AND SOYBEAN. Agronomy Journal, 84, 387-390.

JOHNSON, N. C., GRAHAM, J. H. & SMITH, F. A. 1997. Functioning of mycorrhizal

associations along the mutualism-parasitism continuum. New Phytologist, 135, 575-

586.

JOHNSON, S. N., HAWES, C. & KARLEY, A. J. 2009. Reappraising the role of plant

nutrients as mediators of interactions between root- and foliar-feeding insects.

Functional Ecology, 23, 699-706.

KADAR, I., GULYAS, F., GASPAR, L. & ZILAHY, P. 2000. Mineral nutrition of maize

(Zea mays L.) on chernozem soil I. Novenytermeles, 49, 371-388.

KATOVICH, K., LEVINE, S. J. & YOUNG, D. K. 1998. Characterization and usefulness of

soil-habitat preferences in identification of Phyllophaga (Coleoptera : Scarabaeidae)

larvae. Annals of the Entomological Society of America, 91, 288-297.

KOHL, L., OEHL, F. & VAN DER HEIJDEN, M. G. A. 2014. Agricultural practices

indirectly influence plant productivity and ecosystem services through effects on soil

biota. Ecological Applications, 24, 1842-1853.

LETOURNEAU, D. K. 1986. ASSOCIATIONAL RESISTANCE IN SQUASH

MONOCULTURES AND POLYCULTURES IN TROPICAL MEXICO.

Environmental Entomology, 15, 285-292.

LETOURNEAU, D. K., ARMBRECHT, I., SALGUERO RIVERA, B., MONTOYA

LERMA, J., JIMENEZ CARMONA, E., CONSTANZA DAZA, M., ESCOBAR, S.,

GALINDO, V., GUTIERREZ, C., DUQUE LOPEZ, S., LOPEZ MEJIA, J., ACOSTA

RANGEL, A. M., HERRERA RANGEL, J., RIVERA, L., ARTURO SAAVEDRA,

C., MARINA TORRES, A. & REYES TRUJILLO, A. 2011. Does plant diversity

benefit agroecosystems? A synthetic review. Ecological Applications, 21, 9-21.

LI, L., LI, S. M., SUN, J. H., ZHOU, L. L., BAO, X. G., ZHANG, H. G. & ZHANG, F. S.

2007. Diversity enhances agricultural productivity via rhizosphere phosphorus

Side 58 af 60

facilitation on phosphorus-deficient soils. Proceedings of the National Academy of

Sciences of the United States of America, 104, 11192-11196.

LI, L., TILMAN, D., LAMBERS, H. & ZHANG, F.-S. 2014. Plant diversity and

overyielding: insights from belowground facilitation of intercropping in agriculture.

New Phytologist, 203, 7.

LIEBMAN, M. & DYCK, E. 1993. CROP-ROTATION AND INTERCROPPING

STRATEGIES FOR WEED MANAGEMENT. Ecological Applications, 3, 92-122.

LIU, D. Y., ZHANG, W., YAN, P., CHEN, X. P., ZHANG, F. S. & ZOU, C. Q. 2017. Soil

application of zinc fertilizer could achieve high yield and high grain zinc

concentration in maize. Plant and Soil, 411, 47-55.

LIU, N., CHEN, X. Y., SONG, F. B., LIU, F. L., LIU, S. Q. & ZHU, X. C. 2016. Effects of

Arbuscular Mycorrhiza on Growth and Nutrition of Maize Plants under Low

Temperature Stress. Philippine Agricultural Scientist, 99, 246-252.

LOREAU, M. & HECTOR, A. 2001. Partitioning selection and complementarity in

biodiversity experiments. Nature, 412, 72-76.

MALCOVA, R., VOSATKA, M. & ALBRECHTOVA, J. 1999. Influence of arbuscular

mycorrhizal fungi and simulated acid rain on the growth and coexistence of the

grasses Calamagrostis villosa and Deschampsia flexuosa. Plant and Soil, 207, 45-57.

MARTINEZ-ALCANTARA, B., MARTINEZ-CUENCA, M. R., BERMEJO, A., LEGAZ, F.

& QUINONES, A. 2016. Liquid Organic Fertilizers for Sustainable Agriculture:

Nutrient Uptake of Organic versus Mineral Fertilizers in Citrus Trees. Plos One, 11.

MARTINEZ, E., DOMINGO, F., ROSELLO, A., SERRA, J., BOIXADERA, J. &

LLOVERAS, J. 2017. The effects of dairy cattle manure and mineral N fertilizer on

irrigated maize and soil N and organic C. European Journal of Agronomy, 83, 78-85.

MENG, L. B., ZHANG, A. Y., WANG, F., HAN, X. G., WANG, D. J. & LI, S. M. 2015.

Arbuscular mycorrhizal fungi and rhizobium facilitate nitrogen uptake and transfer in

soybean/maize intercropping system. Frontiers in Plant Science, 6.

MIAH, M. A. H., BISWAS, M. M. & MANNAN, A. 1986. EFFECTS OF SOME

INSECTICIDES ON WHITE GRUB CONTROL AND YIELD OF SUGARCANE.

Tropical Pest Management, 32, 338-340.

MONTESINOS-NAVARRO, A., SEGARRA-MORAGUES, J. G., VALIENTE-BANUET,

A. & VERDU, M. 2012. Plant facilitation occurs between species differing in their

associated arbuscular mycorrhizal fungi. New Phytologist, 196, 835-844.

NIELSEN, G. C., PETERSEN, P. H., THORSTED, M. D., VESTERGAARD, A. V.,

MIKKELSEN, M., KNUDSEN, L., JENSEN, J. E. & ERIKSEN, L. B. 2015. MAJS.

OVERSIGT OVER LANDSFORSØGENE SEGES Planter & Miljø.

ORTIZ-TIMOTEO, J., SANCHEZ-SANCHEZ, O. M. & RAMOS-PRADO, J. M. 2014.

Productive activities and management of the milpa in three communities of the

municipality of Jesus Carranza, Veracruz, Mexico. Polibotanica, 173-191.

PEREZ-GARCIA, O. & DEL CASTILLO, R. F. 2016. The decline of the itinerant milpa and

the maintenance of traditional agrobiodiversity: Crops and weeds coexistence in a

tropical cloud forest area in Oaxaca, Mexico. Agriculture Ecosystems & Environment,

228, 30-37.

PINEDA, S., CRUZ, G., VALLE, J., DE LA ROSA, J. I. F., CHAVARRIETA, J. M.,

ORDONEZ-RESENDIZ, M. & MARTINEZ, A. M. 2012. Arthropod Abundance in

Two Maize Fields in Western Central Mexico. Journal of the Kansas Entomological

Society, 85, 340-352.

PLANTEAVL, D. L. L. 2003. Dyrkningsvejledning: Silomajs.

Side 59 af 60

POSTMA, J. A. & LYNCH, J. P. 2012. Complementarity in root architecture for nutrient

uptake in ancient maize/bean and maize/bean/squash polycultures. Annals of Botany,

110, 521-534.

QIN, H., LU, K., STRONG, P. J., XU, Q., WU, Q., XU, Z., XU, J. & WANG, H. 2015. Long-

term fertilizer application effects on the soil, root arbuscular mycorrhizal fungi and

community composition in rotation agriculture. Applied Soil Ecology, 89, 35-43.

RADCLIFFE, J. E. 1971. EFFECTS OF GRASS GRUB (COSTELYTRA-ZEALANDICA

WHITE) LARVAE ON PASTURE PLANTS .2. EFFECT OF GRASS GRUBS AND

SOIL MOISTURE ON PERENNIAL RYEGRASS AND COCKSFOOT. New

Zealand Journal of Agricultural Research, 14, 607-+.

RAVNSKOV, S. & LARSEN, J. 2007. Mykorrhiza i økologisk planteproduktion. Økologisk

planteavlsberetning. Institut for plantebeskyttelse og skadedyr.

REDECKER, D., KODNER, R. & GRAHAM, L. E. 2000. Glomalean fungi from the

Ordovician. Science, 289, 1920-1921.

RISCH, S. 1980. THE POPULATION-DYNAMICS OF SEVERAL HERBIVOROUS

BEETLES IN A TROPICAL AGROECOSYSTEM - THE EFFECT OF

INTERCROPPING CORN, BEANS AND SQUASH IN COSTA-RICA. Journal of

Applied Ecology, 17, 593-611.

ROUPHAEL, Y., CARDARELLI, M. & COLLA, G. 2015. Role of arbuscular mycorrhizal

fungi in alleviating the adverse effects of acidity and aluminium toxicity in zucchini

squash. Scientia Horticulturae, 188, 97-105.

SABIA, E., CLAPS, S., MORONE, G., BRUNO, A., SEPE, L. & ALEANDRI, R. 2015.

Field inoculation of arbuscular mycorrhiza on maize (Zea mays L.) under low inputs:

preliminary study on quantitative and qualitative aspects. Italian Journal of

Agronomy, 10, 30-33.

SAEKI, K. 2011. Rhizobial measures to evade host defense strategies and endogenous threats

to persistent symbiotic nitrogen fixation: a focus on two legume-rhizobium model

systems. Cellular and Molecular Life Sciences, 68, 1327-1339.

SAPKOTA, P. 2006. White Grub (Phyllophaga spp.) 14.

SHENEFELT, R. D. & SIMKOVER, H. G. 1951. INSECTICIDES FOR CONTROL OF

WHITE GRUBS. Journal of Economic Entomology, 44, 359-362.

SMITH, B. D. 2006. Eastern North America as an independent center of plant domestication.

Proceedings of the National Academy of Sciences of the United States of America,

103, 12223-12228.

SMITH, F. A. & SMITH, S. E. 2011a. What is the significance of the arbuscular mycorrhizal

colonisation of many economically important crop plants? Plant and Soil, 348, 63-79.

SMITH, S. E. & SMITH, F. A. 2011b. Roles of Arbuscular Mycorrhizas in Plant Nutrition

and Growth: New Paradigms from Cellular to Ecosystem Scales. In: MERCHANT, S.

S., BRIGGS, W. R. & ORT, D. (eds.) Annual Review of Plant Biology, Vol 62.

SONG, B. Z., ZHANG, J., WIGGINS, N. L., YAO, Y. C., TANG, G. B. & SANG, X. S.

2012. Intercropping With Aromatic Plants Decreases Herbivore Abundance, Species

Richness, and Shifts Arthropod Community Trophic Structure. Environmental

Entomology, 41, 872-879.

SOUSA, C. D., MENEZES, R. S. C., SAMPAIO, E., OEHL, F., MAIA, L. C., GARRIDO,

M. D. & LIMA, F. D. 2012. Occurrence of arbuscular mycorrhizal fungi after organic

fertilization in maize, cowpea and cotton intercropping systems. Acta Scientiarum-

Agronomy, 34, 149-156.

Side 60 af 60

STATISTIK, D. 2016. DET DYRKEDE AREAL EFTER OMRÅDE, ENHED OG AFGRØDE

[Online]. www.statistikbanken.dk: Danmarks Statistik. [Accessed 22-05 2017].

STERN, W. R. 1993. NITROGEN-FIXATION AND TRANSFER IN INTERCROP

SYSTEMS. Field Crops Research, 34, 335-356.

STEVENS, G. N. & JONES, R. H. 2006. Patterns in soil fertility and root herbivory interact

to influence fine-root dynamics. Ecology, 87, 616-624.

STEVENS, G. N., PIERSON, D. R., NGUYEN, K. & JONES, R. H. 2007. Differences

between resource patches modify root herbivore effects on plants. Plant and Soil, 296,

235-246.

SUBRAMANIAN, K. S. & CHAREST, C. 1995. INFLUENCE OF ARBUSCULAR

MYCORRHIZAE ON THE METABOLISM OF MAIZE UNDER DROUGHT

STRESS. Mycorrhiza, 5, 273-278.

TIMEANDDATE.COM. 2017. https://www.timeanddate.com/sun/mexico/morelia [Online].

[Accessed 31-03 2017].

TRENBATH, B. R. 1993. INTERCROPPING FOR THE MANAGEMENT OF PESTS AND

DISEASES. Field Crops Research, 34, 381-405.

TSUNODA, T., SUZUKI, J.-I. & KANEKO, N. 2017. Fatty acid analyses to detect the larval

feeding preferences of an omnivorous soil-dwelling insect, Anomala cuprea

(Coleoptera: Scarabaeidae). Applied Soil Ecology, 109, 1-6.

VESTERAGER, J. M., NIELSEN, N. E. & HOGH-JENSEN, H. 2008. Effects of cropping

history and phosphorus source on yield and nitrogen fixation in sole and intercropped

cowpea-maize systems. Nutrient Cycling in Agroecosystems, 80, 61-73.

VILLALOBOS, F. J. 1999. The sustainable management of white grubs (Coleoptera :

Melolonthidae) pest of corn in "El Cielo" Biosphere Reserve, Tamaulipas, Mexico.

Journal of Sustainable Agriculture, 14, 5-29.

WALTER, L. E. F., HARTNETT, D. C., HETRICK, B. A. D. & SCHWAB, A. P. 1996.

Interspecific nutrient transfer in a tallgrass prairie plant community. American Journal

of Botany, 83, 180-184.

WEAVER, J. E. & BRUNER, W. E. 1927. Root Development Of Vegetable Crops, London,

Mcgraw-Iill Book Co.

ZHANG, C. C., POSTMA, J. A., YORK, L. M. & LYNCH, J. P. 2014. Root foraging elicits

niche complementarity-dependent yield advantage in the ancient 'three sisters'

(maize/bean/squash) polyculture. Annals of Botany, 114, 1719-1733.

ZHAO, R. X., GUO, W., BI, N., GUO, J. Y., WANG, L. X., ZHAO, J. & ZHANG, J. 2015.

Arbuscular mycorrhizal fungi affect the growth, nutrient uptake and water status of

maize (Zea mays L.) grown in two types of coal mine spoils under drought stress.

Applied Soil Ecology, 88, 41-49.

ZITLALPOPOCA-HERNANDEZ, G., NAJERA-RINCON, M. B., DEL-VAL, E.,

ALARCON, A., JACKSON, T. & LARSEN, J. 2017. Multitrophic interactions

between maize mycorrhizas, the root feeding insect Phyllophaga vetula and the

entomopathogenic fungus Beauveria bassiana. Applied Soil Ecology, 115, 6.

http://www.statistikbanken.dk/

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