UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE CLUJ-NAPOCA DOCTORAL ... · 2013-06-21 · university of agricultural sciences and veterinary medicine cluj-napoca

UNIVERSITY OF AGRICULTURAL SCIENCES AND

VETERINARY MEDICINE

CLUJ-NAPOCA

DOCTORAL SCHOOL

FACULTY OF AGRICULTUE

__________________________________________________

ABSTRACT OF PHD THESIS

PHD STUDENT: SCIENTIFICAL COORDINATOR: DRD. ING. IOAN AUREL POP PROF. UNIV. DR. IOAN HAŞ

CLUJ NAPOCA

2013

Ing. Ioan Aurel Pop Abstract of PHD Thesis

2

UNIVERSITY OF AGRICULTURAL SCIENCES AND

VETERINARY MEDICINE

CLUJ-NAPOCA

DOCTORAL SCHOOL

FACULTY OF AGRICULTUE

__________________________________________________

ABSTRACT OF PHD THESIS

BEHAVIOR OF CEREAL GRAIN VARIETIES AND

CORN HYBRIDS TO PHYTOPATHOGENIC FUNGI

ATTACK AND THE LEVEL OF FUNGUS AND

MYCOTOXINS INFESTATION IN CENTRAL

TRANSYLVANIAN CLIMATIC CONDITIONS

PHD STUDENT: SCIENTIFICAL COORDINATOR: DRD. ING. IOAN AUREL POP PROF. UNIV. DR. IOAN HAŞ

CLUJ NAPOCA

2013


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CONTENT

Introduction …………………………………………………….….…... 4

CHAPTER I

RESEARCH PURPOSE AND OBJECTIVES ……………….……….. 6

CHAPTER II

MATERIALS AND METHOD OF RESEARCH …………….……….. 9

2.1. Biological material …………………………………………………. 9

2.2. Experimental condition ……………………………………………. 10

2.3. Technology applied in the experimental trials …………..………… 11

CHAPTER III

RESULTS AND DISCUSSION …………………………...………….. 13

3.1. The production capacity of varieties and hybrids studied in two

experimental years ……………………………………….…………….. 13

3.2. Production quality in the two experimental years ………..……….. 15

3.3. Identified fungi and mycotoxins ………………………………….. 19

3.4. Studied cultivars disease resistance …………………………….…. 23

3.5. Correlations between quality, biochemical and microbiological

parameters ……………………………………………………….…….. 24

CHAPTER IV

GENERAL CONCLUSIONS …………………………………………. 27


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INTRODUCTION

Given current demographic explosion, getting better quality food without

natural or synthetic contaminants - is an important task in order to reduce human

diseases and complying food security rules due the fact that the levels of

pollutants in the environment is still very high, resulting in ruthless exploitation of

the planet. This situation is similar to livestock production - a source of high

quality food for humans, who consume 70% of the biomass produced in the world

which is why protecting the food chain becomes a necessity.

Difficulties witch persist in technologies for production, harvesting,

storage and conservation of fodder favors degradation of a large proportion of

biomass, especially by installing a wide range of molds, from the least harmful to

those who produce mycotoxins high degree of pathogenicity.

Even "the daily bread" which is the main food for the major part of the

population containing a large amount of the necessary protein, energy and

minerals, unfortunately without realizing presents the risk of entering into the

body of secondary metabolites produced by molds that can be installed on grains.

Undesirable effects of these mycotoxins do not manifest immediately, but only in

cases of severe poisoning, they caused damage in human and animal health

through a process of gradual accumulation. The impact of interactions between

mycotoxins and body is very complex and difficult to quantify going up to the

genome by distorting DNA and because their low molecular mass, some

mycotoxins may go through all food chain: feed, animal products and the final

consumer.

Discussions on the mycotoxins need to have their starting point on cultivars

that are genetically more or less resistant/tolerant to fungal attack during the

growing season and storage.

In this context, Research and Development Station for Cattle Breeding

from Târgu Mureş, University of Medicine and Pharmacy Târgu Mureş and SC

Transapicola SRL have proposed through a research project to approach the

interdisciplinary problems and consequences of fungal pollution on biological

systems, exploring the limiting possibilities of their potential threat and

transmission along the food chain.

Entitled work: ”Behavior of cereal grain varieties and corn hybrids to

phytopathogenic fungi attack and the level of fungus and mycotoxins infestation

in central Transylvanian climatic conditions” includes a number of bibliographic

data combined with own researches, a foray into the world of fungi and


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mycotoxins, presenting european and global rules on permissible quantities of

mycotoxins in food and feed; presentation of methods for identification and

quantification of fungi in order to decipher the taxonomic description of this

paralleled universe of "mold flowers", monitoring the behavior of key grain

varieties and corn hybrids grown in Central Transylvania and detection for the

main types of mycotoxins: aflatoxin, ochratoxin, zearalenone, deoxynivalenol

using the most modern and original means of detection, determination of the

chemical composition, the establishment of correlations between the main

parameters determined, all contained in a “Comprehensive program to prevent

and combat fungi infestation in grain and forage in order to ensure consumer

welfare and protection”.

This paper is neither a beginning nor an end - is a call to new researches

in a world where the oldest living creators on Earth make their way to survive,

and we, the human race, do not fit any of these competitors.

The thesis is divided into six chapters, which comprise 66 tables, 89

figures and 39 conclusions. The bibliography contains a number of recent

nationally and internationally references. Iconography, especially the original

pictures complement the experimental field work.

In carrying out the work, I have benefited by competent and demanding

guidance of Mr. prof. dr. Haş Ioan, to whom I own respectful thanks, profound

gratitude and consideration.

I also thank the Research and Development Station for Cattle Breeding

from Târgu Mureş team, Mr. dr. Cornel Podar for his contribution to the synthesis

of materials and relation between phenomena, Mrs. Justina Lobontiu for

professional support in the field of specialty, Mr. Alin Daniel Gulea for his

collegial support, Mr. dr. ing. Oroian Ioan - ASAS correspondent member - for

assuring all necessary research conditions. Special thanks to Miss biologist

Daniela Ioana Ţerbea for her support, patience in correcting the text and

arrangement of all the materials.

Also thanks to Mr. dr.m.v. Dan Iosif of SC Transapicola SRL for good

collaboration and the collaborators from the University of Medicine and

Pharmacy Târgu Mureş, where they performed biochemical and microbiological

analyzes, especially to Mr. prof. dr. Curticăpean Augustin and Mrs. dr. ing.

Felicia Toma.

I thank family to it’s support.

The Author


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CHAPTER I

RESEARCH PURPOSE AND OBJECTIVES

A primary goal of the food industry is to supply the population with high

quality and healthy food. To achieve this goal, the European Union requires to all

member states compliance to strongly and severe quality standards stipulated in

ratified international agreements, highlighting risk factors, risk assessment and

presentation of all these factors to the society. In this way, manufacturers must be

concerned about the quality of food and on the other hand, the government should

protect health and welfare of consumers and the interests of honest producers,

processors and sellers against unfair competition (MĂRUŢOIU ŞI TOFANĂ,

2005).

Given the existing pathogen potential throughout the food chain, food

quality monitoring at every stage of the production process in order to build a

database allowing us to elaborate antifungal strategies adapted to local conditions

is a priority of the current scientific community.

Thus, a study performed by MARIA ŞERDARU (2010) aimed to achieve a

mycotoxicological screening which led to the analysis of 39 samples from

different feed (grains, fiber, legumes). The content of the main mycotoxins

(aflatoxins, ochratoxin, zearalenone, fumonisins, deoxynivalenol, T-2 toxin) was

determined by ELISA method. The results indicated exceeding maximum limits

for zearalenone content in 69.23% of samples, respectively aflatoxins, ochratoxin,

toxin T-2 to 7.69% of the samples.

In another study, 74 samples of cereals (45 corn samples, 17 wheat

samples, 8 samples of barley, oats 4 samples) from southwestern Romania, were

analyzed in terms of mycological and mycotoxicological parameters (ISRAEL,

2010). Species of fungi, the most common, belonged to the genera Aspergillus

and Fusarium. The most common toxigenic species identified were: A. flavus, A.

ochraceus, A. niger, A. fumigatus, F. graminearum, F. culmorum and F.

verticillioides. Aflatoxin B1 (AFB1), deoxynivalenol (DON), zearalenone (ZEA),

fumonisins (FUMO) and ochratoxin A (OTA) were determined by ELISA

method. More than 90% of the samples were contaminated with at least one of the

examined mycotoxins. Among the analyzed grains, corn is most strongly affected

by fungal infestation and mycotoxin contamination. Of the total 84.21% of

samples contaminated with aflatoxin are samples of corn. Ochratoxin A was


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recovered only at the rate of 14.86% in corn. DON, ZEA and FUMO has been

found in the maize at a rate of 47.36%, 83.33% and 39.13% of the samples

contaminated with these mycotoxins.

Determined concentrations match within EU norms, except for aflatoxin

where 18.75% of the samples exceed the maximum levels allowed by EU rules.

The results show that the cereals in southwestern Romania are often

contaminated with fungi and mycotoxins and requires strict control of grain

(TABUC, 2010).

As mentioned prevention of mycotoxins is possible through three very

important ways:

- Creating cultivars resistant to diseases

- Agro-technical measures: crop rotation, soil tillage system, fertilization,

varieties choice and plant protection treatments (diseases, pests, weeds, etc.).

- Ensure proper storage conditions (PASCU, 2008).

Discussions on the mycotoxins subject have to have the starting point on

cultivars that are genetically more or less resistant/tolerant to fungal attack during

the growing season and storage.

Although there are a lot of difficulties phytopathology research programs

and plant improvement/breeding taking place in Romania and abroad have

intensified in recent years by testing the resistance to diseases in order to create

genetic material with a high level of tolerance to Fusarium (ITTU, 2008).

Determining the risk of fungi and mycotoxin contamination of grain

production from Transylvania, we have to take into account the range of varieties

of cereals grown in the area, their resistance to pests and diseases and their level

of contamination with mycotoxins (aflatoxin, deoxynivalenol, zearalenone,

ochratoxin).

In this paper we plan to realize a complex approach to the interdisciplinary

problems and consequences generated by fungal diseases on biological systems,

limiting their opportunities and the threat potential on the food chain.

The purpose of this paper is to monitor the behavior of several varieties of

cereals and commercial corn hybrids in terms of resistance to diseases and their

impact on grain quality in the natural conditions existing in central Transylvania.

Objectives that lead to the goal of the paper are:

- a bibliographic study on the implications of fungi and mycotoxins in

agriculture;

- monitoring the behavior of some cereals varieties and corn hybrids

regarding disease resistance in experimental field trials;

- increase of grain production and quality by promoting the cultivation of

tolerant to disease varieties and optimized crop protection technology;


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- monitoring and evaluation of the chemical composition of several cereal

varieties and the achievement of high precision biochemical analysis,

microbiological and chemical analysis;

- establishment of some correlations between studied parameter values in

order to know the degree of association between them;

- reduction of fungal pollution for feed and food;

- effective dissemination of the results so that each segment of the market

(producers, processors, consumers) can use this research results.

The interdisciplinary nature of the thesis calls for knowledge from: plant

protection, genetics and breeding, plant growing, mycology, mycotoxicology,

biochemistry fields.


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CHAPTER II

MATERIALS AND METHOD OF RESEARCH

2.1. BIOLOGICAL MATERIAL

Recent studies have shown the existence of high concentrations of

mycotoxins in cereals and cereal products (ISRAEL,2010; TABUC 2010; ITTU

2008, POP SI COLAB, 2010). These results requires a vigilance attitude and a set

of measures in order to protect consumers and to increase confidence in the

products.

Knowledge of grain biological particularities is an essential step in the

process of plant breeding and in researches for cultivation technology which aims

to obtain high yields and healthy for consumption products.

For this purpose at SCDCB Mureş in 2008-2010 two experiments were

settled aiming to observe the behavior of some cereals and corn varieties at

phytopathogenic fungi attack and the degree of infection with fungi and

mycotoxins in natural climatic conditions from central Transylvania.

In order to choose the most tolerant cereals genotypes to diseases we used

data from research station records.

At wheat crop trials, besides the three zoned varieties Arieşan, Apullum

and Ardeal 1 we introduced newer Romanian varieties: Magistral and Gasparom

created at SCDA Suceava, a line created in Turda and two foreign varieties,

Renan and Exotic. In the experimental field of the year 2009-2010 due to

increasing interest by farmers in the experiment were introduced varieties: Glosa

and Dumbrava creations of INCDA Fundulea and SCDA Turda.

Triticale has established itself as a valuable plant, because of the high

production capacity and high-protein substances, being used in animal feeding.

Varieties tested (six in number) are creations obtained at INCDA Fundulea.

Because the high cost of biochemical analysis we reduced the number of triticale

varieties studied in 2009-2010, the biochemical analysis not being done for:

Trilstar, Style, 0474T1 varieties. In turn, was introduced in the experiment Haiduc

variety. In 2008-2009 trials, wheat and triticale cultivars include three varieties of

barley with six rows, two of which are of foreign origin - Gerlac and Plaisant -

and one Romanian - Regal.


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At maize biological material tested in 2008 was represented by 24 hybrids

of different origin: SCDA Turda (E) -5, Pioneer (U.S.) -8, NK Syngenta (FR) -4,

Limagrain (FR) -2 and RodBun (A) -5. As it is obvious our aim is to test hybrids

cultivated for a long time in the area (Turda, Pioneer) and also the newly one

introduced in the area (Limagrain, RodBun, Syngenta). Biological material

selection was done based on farmers' preferences.

In 2009, the biological material consisted of 24 maize hybrids including 3

created at SCDA Turda, 8 Pioneer creations, 6 KWS creations, 1 LG creation, 4

Rod Bun creations.

2.2 EXPERIMENTAL CONDITION

Trials were carried out in the Borssofold experimental field belonging to

Research and Development Station for Cattle Breeding Târgu Mureş being made

by researchers from the department of plant production research.

Year 2008 was a hot year in which the annual average temperature

exceeded the average temperature for the last 113 years with 2.3 Celsius degrees.

In terms of rainfall year is characterized by a deficiency of precipitation of

113l/mp.

The contribution of rainfall between April 1 to August 31 was 304.3 mm,

with a deficit of 78.4 mm from the annual average. The small amount of rainfall

was recorded in April, only 3 mm in comparison with 49 mm - the annual

average of the month. his very low quantities affected corn emergence.

The climate conditions of the agricultural year 2008-2009 were

characterized in terms of heat by overcoming the multiannual average from

October 2008 till August 2009 with 1.6 º C, the highest monthly average overrun

occurred in April and July, with 3.0 º C respectively 3.3 º C.

In terms of rainfall contribution, during the period October 2008 -

10.07.2009 there was a deficit of -142 mm, the largest deficit was registered in

April -47.8 mm and may -24.2 mm.

Rainfall deficiency for April and May overlapped with high thermal

regime, influenced plant growth in the first part of vegetation.

In terms of heat the spring of 2009 started with temperatures above the

annual average 3 º C April, 1.1 º C in May, and the rainfall was deficient with of -

47.8 mm in April and 24.2 mm in May. Precipitation deficit throughout the

growing season (1.05-30.09) crop totaled -159.7 mm.

Critical periods appeared at emergence and flowering. Sum of active

temperatures recorded in the range 15.04-30.09 totaled 1365.5º C. Although

emergence was delayed due to lack of water in April, the amount of heat of


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coming months created thermal conditions for all hybrids to reach technical

maturity till 30.09.2009.

Figure 2,1, - Temperature and rainfall during 2009

Year 2009 - 2010 in Târgu Mureş is characterized as a rainy year, very

rainy during May, June and excessively wet and very dry in July. In terms of

thermal parameters mount May was normal June and July were hot and warm

(Figure 1.2). In terms of temperature and rainfall, the year is characterized by an

average annual temperature of 10 ° C, compared to the annual average of 8.6° C

with a positive deviation of 1.4 ° C and annual rainfall amount was 667 mm, close

to yearly average amount of 622 mm, with a positive deviation of 45 mm.

2.3. TECHNOLOGY APPLIED IN THE EXPERIMENTAL TRIALS

For the cereals trials tillage works consisted of a stubble disc furrow,

followed by plowing at a dept of 20 cm and two works with disc harrow for

seedbed preparation. At the seedbed preparation 80 kg NPK was applied to,

taking into account the preceding crop (spring vetch). Sowing was done with

experimental field (SCE 8), 8 rows seed drill at 12.5 cm distance between rows on

a length of 8.5 m/repetition.

Corn crop was placed after wheat in a 3-year rotation (wheat, corn, beans).

Soil tillage consisted of a disc in August, deep plowing in October, spring disking

and leveling and a work with combinator for seedbed preparation and also for

fertilizer and herbicide incorporation.


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Throughout the growing season we monitored the influence of climatic

factors on plant development, the varietal resistance to stress and a particular

emphasis was made on cultivars natural resistance to foliar and ear diseases.

In winter crops, the main foliar diseases present in the area are produced

by: Erysiphe graminis, Septoria sp., Puccinia spp, Fusarium ssp. and

Helminthosporium spp at barley. Phenotypic observations were made with grades

varying from 1-9 for each disease, note 1 characterizing a minimal attack and the

note 9 represents a heavy attack with a very high frequency.

Cereal harvest was done in two stages: reaping and threshing, using an

experimental thresher. At harvest were determined: total production in bundles,

grain yield, moisture and frequency of Fusarium spp. attack on grains, MMB

determination was done in the laboratory.

At the corn crop besides the usual records and observations (date of

sowing, time of emergence, flowering date, physiological maturity, aspect of the

plot) were also made observations on disease resistance.

The main targeted diseases were: rusts, helmintosporium disease, corn

smut, fusarium blight and especially fusarium on cobs. The attack was determined

by phenotypic observations with grades between 1 and 9 for each disease, note 1

characterizing a minimal attack and the note 9 represents a heavy attack with a

very high frequency. For corn smut were counted affected plants and the

Fusarium on cobs were counted the affected cobs.

At harvest were determined: grain yield, fusarium affected cobs frequency,

moisture at harvest.

Harvesting was done manually using electronical weighter and moisture

meter for field determinations.

All samples from experimental field were analyzed for chemical

composition by an Instalab 610 infrared analyzer that allows the determination of

protein, oil, starch, fiber, ash and gluten.

Samples from the field were sent to University of Medicine and Pharmacy

Târgu-Mureş, National Research and Development Institute for Biology and

Animal Nutrition Baloteşti to carry out microbiological and biochemical

determinations. The main post-harvest determinations were:

- Microbiological analysis in order to identify and quantify the species of

fungi;

- Biochemical analysis performed to identify and quantify the main types

of mycotoxins;

- Chemical analysis (content of protein, starch, ash, fiber, oils).


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CHAPTER III

RESULTS AND DISCUSSION

Specialists has always been concerned towards the achievement of

increased production per unit and to obtain appropriate quality agri-food

products.

The objectives to achieve the purpose of this thesis are:

- Identify the main characteristics of the cereals and corn hybrids that are

the subject of this work;

- Determination the production capacity for wheat varieties and corn

hybrids studied;

- Tracking behavior on disease resistance of varieties of cereals and maize

hybrids in yield trials;

- Monitoring and evaluation the chemical composition to determine the

quality of varieties and hybrids;

- Achieving high precision and biochemical analysis and microbiological

analysis;

- Regression analysis between various indicators of quality,

microbiological and biochemical indices in order to establish the relationship

between these factors.

3.1. THE PRODUCTION CAPACITY OF VARIETIES AND

HYBRIDS STUDIED IN TWO EXPERIMENTAL YEARS

Average yield of the seven wheat varieties tested in both experimental

years was 6930 kg/ha, with only 89.4 kg/ha higher in 2008-2009 crop year than

the 2009-2010.

Looking at the average production in the two experimental years

(Figura3.1) the highest yield obtained Gasparom variety, exceeding the control

variety (Arieşan) with 446 kg/ha. Ardeal variety obtained the smallest production

with 9.57% which is lower than the Arieşan variety. Other varieties have obtained

productions between 92.46% and 97.84%, compared to the control variety that

was 7171 kg/ha.

The triticale species differences production were + 3185 kg/ha for Titan

and 1755 kg/ha for Plai in 2008-2009, compared to 2009-2010, the difference


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between the average yield was 2469 kg/ha. Control variety Plai recorded an

average production over the two years of experience of 6920 kg/ha and 6380

kg/ha Titan cultivar.

Figure 3.1.- Wheat average yields recorded in the two experimental years

In the agricultural year 2008-2009 were tested three varieties of barley,

two of which are of foreign origin. The yields achieved were between 6189 kg/ha

and 6683 kg/ha, lower than the species of wheat and triticale. The lowest yield

was obtained from cultivar Plaisant, 6189 kg/ha significantly negative production.

In maize averaged production STAS for two experimental years (Table

3.1.) varied between 6692 kg/ha and 10337.5 kg/ha, the maximum production

obtained PR39F58 hybrid, exceeding the production of control variety 2596.5

kg/ha (33.5 %), making a very significant positive production.

Distinct significant positive productions have achieved Turda Star,

PR38A24, Loretto, LG 3330, Monallisa and PR39D81, exceeding the control

variety with percentages ranging between 15 and 20%.


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Table 3.1.

Average production of corn obtained in comparative trials, Tg-Mureş, 2009- 2009

No. Variants Average

(kg/ha)

(%)

Differance

(kg/ha) Semnification

1 T 201 7741 -

2 T 200 6692 86.41 -1049.00 o

3 PR38A24 9268 119.7 1527.00 **

4 T Star 9090 117.4 1349.00 **

5 Atalante 8626.5 111.4 885.50 *

6 Loretto 9180 118.6 1439.00 **

7 LG 3330 9340.5 120.7 1599.50 **

8 Monalisa 8961.5 115.8 1220.50 **

9 PR39D81 8977.5 116 1236.50 **

10 Clarica 9562.5 123.5 1821.50 ***

11 PR39F58 10337.5 133.5 2596.50 ***

DL 5%=870.52, DL 1% =1182.9, DL 0,1%= 1603.6

3.2. PRODUCTION QUALITY IN THE TWO EXPERIMENTAL

YEARS

Quality analysis were performed using an infrared analyzer type 610

Instalab purchased by SCDCB Mureş and calibrated for the determination of

quality parameters in the tables below.


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Table 3.2.

Wheat, triticale and barley kernels chemical content obtained from comparative

trials, Tg-Mureş, 2008-2009

Variety Protein

(%)

Oil

(%)

Starch

(%)

Fibre

(%)

Ash

(%)

Gluten

(%)

Wheat

Arieşan 10.8 1.45 52.3 2.85 1.8 21.0

Ardeal 9.8 1.33 52.1 3.02 1.7 18.8

Magistral 10.5 1.62 51.1 3.07 2.3 20.5

Renan 11.9 1.46 49.9 3.14 2.2 24.4

Exotic 9.5 1.30 51.6 2.99 1.4 19.1

Gasparom 9.2 1.21 51.1 3.09 1.6 18.8

Turda2000 9.4 1.24 52.8 2.91 1.5 18.0

Apullum 10.5 1.26 50.8 3.10 1.8 21.2

Triticale

Plai 10.15 2.78

Titan 10.3 2.26

Trilstar 8.85 3.01

Stil 10.55 2.725

00474T1-1 10.5 1.7

Barley

Gerlac 9.25

Regal 9.4

Plaisant 9.15


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Table 3.3.

Wheat and triticale kernels chemical content obtained from comparative trials,

Tg-Mureş, 2009-2010

Variety Protein

(%)

Oil

(%)

Starch

(%)

Fibre

(%)

Ash

(%)

Gluten

(%) Variety

Wheat

1 Arieşan 13.25 1.425 51.80 2.88 1.975 24.15

2 Apullum 12.30 1.35 51.80 2.92 1.92 24.25

3 Ardeal 12.60 1.315 50.20 3.14 2.15 25.55

4 Gasparom 9.15 1.36 52.25 2.89 1.435 18.15

5 Magistral 10.45 1.38 52.25 2.85 1.54 20.75

6 Glosa 10.85 1.125 50.60 3.12 1.715 22.2

7 T2000 11.85 1.375 52.35 2.81 1.86 23.3

8 Dumbrava 11.80 1.24 51.00 2.96 1.72 24.2

9 Exotic 10.25 1.055 51.30 3.05 1.5 20.8

Triticale

1 Plai 8.85 3.075

2 Titan 10.25 3.06

3 Haiduc 10.00 3.05

From the two tables (Table 3.2 and 3.3) is observed a higher protein

content recorded in 2008-2009 crop year that registered in 2010 all experimental

variants of wheat. Thus the variety with the highest protein content was found to

be control variety Arieşan, it has an average content of protein of 12% in the two

years and was exceeded only by the variety Renan in 2008-2009 that had a

significant positive protein content. The lowest concentration of protein in the two

experimental years had Gasparom variety. Also Exotic variety obtaind lower

concentration of protein.


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The average of the oil content in the two years of the experiment was the

same. Magistral variety in 2008-2009 recorded the highest oil content (1.62%)

followed by Renan it containing a variety with only 4% higher than the control

variety. In 2009-2010 Glosa and Exotic varieties registred very significant

negative values are regarding oil content and Dumbrava variety registred

significantly distinct negative concentrations to control variety who got the

maximum concentration of all variants studied: 1.43%.

Average starch content in the two years of experience was 51.7%, there

were no differences between years. Turda 2000 variety has shown the superiority

in terms of starch content in both crop years gaining an average of 52.58%. In

contrast, the variety with the lowest concentration was Ardeal (51.13%) which in

2009-2010 registered a very significant negative concentration of 50.2%. The

lowest average concentration of starch was recorded by variety Renan in 2008-

2009.

Regarding the fiber content Areal variety registered the higher average

concentrations (distinctly significant and very significant). Fiber concentrations

varied in the two experimental years between 2.86% and 3.08%, the differences

from one year to another is unnoticeable. The lowest average content was

recorded by Arieºan variety (2.86%) and the largest one by Ardeal variety .

Minerals were present in grain content in percentages ranging from 1.4%

(2008-2009 Exotic variety) and 2.3% (Magistral variety, crop year 2008-2009),

the highest average concentration of minerals in the two years of study being

registered by Ardeal variety (1.94%). Significantly distinct negative values

recorded Exotic varieties (1.45%) and Gasparom (1.50%) varieties.

In maize trials - in collaboration with the team of researchers from SCDA

Turda in 2008 were made chemical composition analysis for each experimental

variant, being thus made 72 analysis. Analyses were performed using a NIR

spectrofotometer Instalab 600.

Analyzed for the two experimental years (Table 3.4.) average starch

content varied between 60% and 63.1% highlighting PR38A24 hybrid with a

statistically significant energy content in comparison to control variety.

PR39F58 hybrid recorded the highest average oil content of the two-year

study of 5%, followed by hybrid Turda 201 containing 4.9%.


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Table 3.4.

Corn kernels chemical content obtained from Tg-Mureş trials 2008 and 2009

Hybrid Starch

(%)

Oil

(%)

Protein

(%)

Fibre

(%)

Ash

(%)

T 201 60.9 4.9 9.3 4.9 3.3

T 200 60.0 4.5 10.9 5.0 3.8

PR38A24 62.2 4.7 9.5 5.2 2.6

T Star 60.7 4.9 9.7 5.2 2.8

Atalante 61.7 4.6 9.2 4.3 3.8

Loretto 61.6 4.5 9.2 4.2 3.6

LG 3330 63.1 4.0 10.5 4.7 2.4

Monalisa 61.7 4.5 9.8 5.0 2.5

PR39D81 62.1 4.6 9.7 5.2 2.6

Clarica 61.7 4.4 9.9 4.4 3.1

PR39F58 61.4 5.0 9.6 5.5 2.8

Sursa: Original

The average protein content detected in the two experimental years in the

chemical composition of maize kernels ranged between 9.2% at Loretto hybrid

and 10.9% at hybrid Turda 200. Turda 200 had the highest protein level.

The average calculated content of fiber on the two years of experience

ranged from 4.2% to 5.5%.

The highest average ash content in the two experimental years was

achieved by hybrid Atalante 3.8%, followed by hybrid Turda 200, both hybrids

registring significantly positive average concentrations.

3.3. IDENTIFIED FUNGI AND MYCOTOXINS

Biochemical tests in order to detect target mycotoxins were conducted in

collaboration with the University of Medicine and Pharmacy, Department of

Biochemistry, Centre for Public Health Mureº using HPLC methods. In 2009 all

biochemical analysis were carried out at INCDBNA Baloteºti by ELISA method.


20

At cereals in 2008-2009 the highest load of fungi was present in Stil

variety of triticale (11237 CFU/g). Analysing the overall fungal load can be

observed the presence of high-toxigenic fungi such as Penicillium spp - 73.4%,

Aspergillus fumigatus - 14% and Fusarium spp. 6.6%, the remaining fungies

showing the average load of less than 3%.

Figure.3.2.- Fungus yield and type from cereals harvested in 2009

As can be seen (Table 3.5.) in the two years of experimentation

zearalenone concentrations are within the maximum imposed limits by European

Regulations. Exotic variety is approaching this limit, however, the average

concentration being 94.33 ng/g, significantly positive value. The lowest

concentration of zearalenone is obtained from the Ardeal variety which is 61.5%

of that of the control variety. Triticale varieties studied had values that fall within

the parameters of law.


21

Tabel 3.5./ Table 3.5.

Average zearalenone concentration obtained during the two years of trial

No Variants Average

(ng/g)

Differance

(ng/g)

Relative

(%) Semnification

1 Arieşan 29.17

2 Ardeal 17.93 -11.24 61.5

3 Magistral 35.69 6.52 122.4

4 Exotic 94.33 65.16 323.4 *

5 Gasparom 21.41 -7.76 73.4

6 Turda

2000 39.46 10.29 135.3

7 Apullum 50.66 21.49 173.7

DL 5% = 49.20, DL 1%=69.07, DL 0.1% = 97.50

Table 3.6.

Average deoxynivalenol concentration abtained during the two years of trial

No Variants Average

(ng/g)

Differance

(ng/g)

Relative

(%) Semnification

1 Arieşan 108.80

2 Ardeal 63.74 -45.06 58.6

3 Magistral 406.35 297.55 373.5 *

4 Exotic 333.51 224.71 306.5

5 Gasparom 436.48 327.68 401.2 **

6 Turda 2000 595.34 486.54 547.2 ***

7 Apullum 432.21 323.41 397.2 **

DL 5% = 226.25, DL 1%=317.58, DL 0.1% = 448.34


22

Table 3.7 shows the average concentrations of mycotoxins and average

load of fungi obtained by corn hybrids that have been tested both in 2008 and in

2009. The highest calculated average concentration was obtained by Loretto

hybrid whose composition of aflatoxin and ochratoxin exceeds the and 5 ng/g

maximum level set by European legislation. The average concentration of 13.07

ng/g aflatoxin is the highest of all, exceeding the threshold of 5 ng/g by 2.6 times.

Ochratoxin concentration of 5.8 ng/g exceeds the legal limit of 1.2 times.

Table 3.7.

Mycotoxins concentration and fungus load of hybrids studied at Tg-Mureş

No Variants

Afla

toxina

ng/g

Ochra

toxina

ng/g

Zear

alenona

ng/g

Asperg

illus

spp.

Penicil

lium

spp.

Fusar

ium

spp.

1 T 201 3.07 6.7 12.2 2350 23000.0 900.0

2 T 200 4.98 6.5 11.6 2500 18500.0 0.0

3 PR38A24 2.27 10.0 38.9 1250 27000.0 100.0

4 T Star 2.61 11.0 15.5 1500 28500.0 0.0

5 Atalante 10.97 4.3 102.6 6050 500.0 5750.0

6 Loretto 13.07 5.8 19.5 7100 19000.0 3100.0

7 LG 3330 1.77 3.5 37.4 750 9250.0 1500.0

8 Monalisa 3.71 4.5 24.3 2250 500.0 500.0

9 PR39D81 2.62 3.4 93.9 750 2750.0 4150.0

10 Clarica 4.13 6.8 30.5 2300 20500.0 2000.0

11 PR39F58 8.00 4.5 29.8 5750 7500.0 2000.0

Average 5.20 6.08 37.83 2959.09 14272.73 1818.18

Zearalenone concentration did not registered exceeding of the legal

parameters set by Community legislation.

Microbiological analysis reveal high load of the Penicillium genus fungi

present on kernels of corn hybrids derived from the experimental field. Out of

toxigenic fungi belonging to the genus Penicillium we identified 74.92%, 9.54%

belonging to the genus Fusarium and 15.53% to Aspergillus genus.


23

3.4. STUDIED CULTIVARS DISEASE RESISTANCE

Throughout the growing season we monitored the influence of climatic

factors on plant development, varietal resistance to stressors and particular

emphasis was placed on monitoring the major foliar and ear diseases.

In the 2008-2009 agricultural year climatic conditions from spring were

characterized by lack of rainfall and high temperatures witch stopped mildew

attack at the three winter cereal species, however, in spring 2010 the abundance of

precipitation and annual average temperatures favored powdery mildew attack at

the two species of winter cereals studied, the attack being evaluated as

moderately.

Of the wheat varieties, the attack was not present at Ardeal, Magistral and

Gasparom varieties, Exotic and Glosa varieties showed to be sensitive and

Arieşan variety manifested a high sensitivity. In triticale crop attack was not

present.

Septoria is reported as a dominant disease in wheat and triticale crop. It is

the most damaging disease and reducing production by 30-40% in case of

favorable development of the fungus if no action is taken to protect crops.

Although in crop production Arieşan wheat variety is susceptible to this

disease, the attack manifested in the experience made in 2010 was moderate

(grade 5). Best behavior to Septoria had Exotic and Gasparom varieties (Note 2)

and the most sensitive was Arieşan.

Except Gasparom and Exotic varieties who experienced a mild attack of

rust brown all other varieties manifested no attack.

In 2009 the most susceptible to this pathogen were Arieşan and Apullum

wheat varieties.

The following table illustrates the results of observations made from corn

hybrids tested both in 2008 and in 2009.


24

Table 3.8.

Observation regarding hybrids diseases resistance in 2008 and 2009

Resistance

No. Hybrid

Hel

mint

hosp

oriu

m

Pucc

inia

spp.

Fusari

um

spp.

(%)

Ustil

ago

spp.

(%)

Ostrini

a

nubila

lis

(%)

No of

days till

phisiologi

cal

maturity

Dise

ases

Inde

x -

Ib

1 T 201 2.5 3 6 3.8 28.7 113.5 96.4

2 T 200 2.5 2 5 6.8 29.9 115.0 96.1

3 PR38A24 2.5 2.5 9.4 4.1 37.6 110.3 96.0

4 T Star 3.5 3 5.75 1.3 28.6 115.0 96.7

5 Atalante 3 2.5 8.55 0.8 35.8 107.5 96.3

6 Loretto 4 2.5 8.05 4.1 29.3 104.0 95.7

7 LG 3330 2.5 4 6.1 3.3 36.7 112.3 96.1

8 Monalisa 3 2.5 6.8 2.6 33.3 112.0 96.4

9 PR39D81 2.5 2.5 4.9 0.0 40.3 109.8 97.2

10 Clarica 3.5 2.5 7.1 0.8 31.2 111.0 96.6

11 PR39F58 3 2.5 7.95 3.4 36.9 112.5 95.9

Note: Attack intensity: note 1 – minimum attack, note 9 – maxim attack

3.5.CORRELATIONS BETWEEN QUALITY, BIOCHEMICAL

AND MICROBIOLOGICAL PARAMETERS

In order to analyze the association between mycotoxins concentrations of

fungi load and quality parameters studied in the 24 commercial corn hybrids we

calculated the correlation coefficients (r). The study of relationship between these

parameters is of great utility in field practice and animal husbandry by allowing

selection of hybrids possessing simultaneous the combination of several quality

indices, but also draws our attention to the difficulties in choosing the range of

hybrids with resistance to diseases and adaptability to local conditions.


25

Very significant positive correlations were established at interaction

between the protein content and gluten, between fungi of the genus Fusarium and

deoxinivalenol (DON) concentration. A significant negative correlation was

obtained from the analysis of starch content compared to fiber.

Figure.3.3.- Relation between wheat protein content and gluten

Sursa/Source: Original

The association of the protein content with gluten in wheat samples

provided from experimental field is described by a straight ascending slope of the

regression line, which indicates that the gluten content is directly proportional to

the protein (Figure 3.3.). Distribution points around the regression line is highly

clustered, indicating the very close relationship between the two traits analyzed

for both species. The coefficient of determination r2 = 0.93 indicates that variation

gluten is caused in a percent of 93% to the variation of protein content. The

intensity of the relationship between protein content and gluten content was

highlighted by the linear correlation coefficient (r = 0.96), indicating a strong

correlation, therefore the points are clustered around the regression line. Our

results confirm the results obtained from SCDA Turda by Edith Szekely.

The length of the regression line and the distribution of points clustered

around this line show the close relationship existing between the load of fungi of

the genus Fusarium and the concentration of deoxynivalenol (Figure 3.4).

Coefficient of determination R2 = 0.96 obtained show that variation of


26

deoxynivalenol concentration is 96% due the variation of fungi load from the

Fusarium genus. The intensity of the relationship between the number of fungi

from the genus Fusarium and concentration of deoxinivalenol has been observed

by coefficient of linear correlation (r = 98) indicating a very strong bond between

the two variables.

Figure.3.4.-Relation between Fusarium spp. (CFU/g) and deoxynivalenol (DON)

Sursa/Source: Original


27

CHAPTER IV

GENERAL CONCLUSIONS

1. Since the period of growth and development and later during storage

both the vegetative and the final product are constantly assaulted by a variety of

phytopathogenic fungi and molds produced by different species of fungi. Fungi,

by the action of secreted enzymes, break down the nutrients in food and feed they

come into contact. Mycotoxins are toxic secondary metabolites produced by fungi

on different substrates.

2. The main pathogenic fungi that cause significant damage to crops,

converting crops to mycotoxin contamination sources, belong to the following

genera: Fusarium (Fusarium), Tilettia (bunt), Ustilago (embers), Puccinia (rust),

Claviceps purpurea (ergot), Penicillium (mold), Aspergillus (molds).

3. Regulations (EC) no. 1126/2007 and (EC) no. 1881/2006 of the

European Commission sets maximum levels for certain contaminants in

foodstuffs and in the content of mycotoxins. The maximum limits of mycotoxins

in animal feed under these regulations are: 2 ng/g for aflatoxin B1, 5 ng/g for

aflatoxin, 5 ng/g for ochratoxin, 100 ng/g zearalenone for unprocessed cereals and

350 ng/g for zearalenone maize.

4. As mentioned prevention of mycotoxins is possible through three very

important ways: - Creating cultivars resistant to diseases

- Agro-technical measures: crop rotation, soil tillage system, fertilization,

varieties choice and plant protection treatments (diseases, pests, weeds, etc.).

- Ensure proper storage conditions

5. Discussions on the mycotoxins subject have to have the starting point

on cultivars that are genetically more or less resistant/tolerant to fungal attack

during the growing season and storage. With regard to this aspect, in recent years,

scientists in the field of plant breeding have been increasingly focused on the

germplasm derived from wild species and species ancestral use in order to

increase the natural resistance to fungal attack.


28

6. The main autumn crops foliar diseases presented in our area are

produced by: Erysiphe graminis, Septoria spp., Puccinia spp, Fusarium and

Helminthosporium spp. on barley. Mildew and rust attack did not result in the first

year of experience. Arieşan and Exotic varieties showed a higher sensitivity to

powdery mildew, the variety Arieşan showed high sensitivity to Septoria spp. and

Fusarium spp. At triticale the highest tolerance to Fusarium spp. showed Titan

cultivar. The most sensitive barley cultivar to cultivar Helminthosporium shown

to be Gerlac.

7. Looking at the average production for the two experimental years, the

highest yield was obtained by Gasparom variety witch surpassed the control

variety - Arieşan with 446 kg/ha. Variety Ardeal obtained the smallest production,

lower with 9.57% than the variety Arieşan. Other varieties have obtained yields

between 92.46% and 97.84% of the control variety witch was 7171 kg/ha.

8. The protein content recorded in the 2008-2009 crop year was higher

than the one registered in 2010 for all experimental variants of wheat. Thus the

variety with the highest protein content was found to be variety Arieşan with an

average of 12% protein content for two experimental years. This content was

exceeded only in 2008-2009 by Renan variety witch had a significantly higher

protein content.

9. During 2009-2010 the average content of protein in fertilized field was

with 24% higher than the control variant (not fertilized), the content of protein

being highly correlated with the gluten.

10. In the two years of experiments average concentrations of zearalenone

are within the maximum limits imposed by European regulations. Exotic variety

is approaching this limit, however, the average concentration was 94.33 ng/g,

significantly positive value. The lowest concentration of zearalenone is obtained

from the strain Ardeal which is 61.5% of that of the control variety.

11. Comparing agrofunds, wheat average concentration of zearalenone in

the fertilized field is 60 ppb and in not fertilized system is 71 ppb.

12. For Triticale crop the average zearalenone concentration on fertilized

environment is 145.5 ppb while in the not fertilized environment is 192.5 ppb, so,

the content of zearalenone was higher for variants from the not fertilized system.


29

13. The average deoxynivalenol concentrations does not exceed the limit of

1,250 ng/g set by European legislation. The highest value very significantly

positive, was achieved by Turda 2000 variety which was followed by Gasparom

variety (436.48 ng/g) and Apullum variety (432.21 ng/g).

14. Despite the favorable conditions of disease development in the

agricultural year 2009-2010, none of the samples showed concentrations above

the maximum level of deoxynivalenol set by law, maximum concentration was

recorded at Dumbrava variety fertilized, average DON concentrations for all

samples being around 550 ppm. The average concentration of DON for fertilized

agrofunds is about 500 ppb, whereas the average concentration of DON for not

fertilized agrofunds was 590 ppb.

15. Analyzing global fungal load we can observe the presence of toxigenic

fungi Penicillium spp -27.6%, Aspergillus fumigatus - 14% and 57.8% Fusarium

spp.

16. Number of samples chemistry, biochemistry and microbiology

analyzed have allowed the development of mathematical correlations:

- very significant positive correlation has been established as a result of

the interaction between the protein content and gluten (R2 = 0.93, r = 0.96) and

the load of Fusarium genus fungi with the concentration of deoxynivalenol (R2 =

0.96, r = 0.98). A significant negative correlation was obtained from the analysis

of starch content compared to the fiber (R2 = .87).

- distinct significant positive correlations were established by the

interaction between the ash content and gluten, oil and ash, Aspergillus fungi load

with Penicillium and Fusarium fungus load.

17. The average starch content of corn ranged between 60 and 63.1%

highlighting the distinct significant PR38A24 hybrid as having high energy

content. Lower concentrations showed that hybrids created at SCDA Turda: Turda

200, Turda Turda 201 and Star.

18. The average protein content of the two experimental years ranged from

9.17% (hybrid Loretto) and 10.88% Turda 200 hybrid witch had the highest

protein level. Significant positive protein content showed also LG 3330hybrid.

19. The average content of fiber calculated for the two years of

experiments ranged between 4.2% and 5.47%. The lowest content was recorded in


30

hybrid Loretto, significant negative concentrations occurred at Atalante and

Clarica hybrids. The highest average fiber content was obtained by PR39F58

hybrid.

20. Of the 11 hybrids tested in the two years of experiments, only four of

them are within the legal limits established by European regulations for

mycotoxins. Average ochratoxin concentration is 6.08 ng/g exceeding 1.3 times

the legal maximum allowed limit. The highest mean concentration of ochratoxin

was obtained by Turda Star hybrid respectively 10.97 ng/g The highest average

concentration of 13.07 ng/g aflatoxin was determined at Loretto hybrid.

21. Zearalenone concentration complies within legal parameters, no

exceeding of the allowed limits set by EU legislation were registered.

22. Microbiological analyzes reveal high load of the fungi from

Penicillium genus present on corn hybrids kernels derived from the experimental

field. Out of toxigenic fungi 74.92% belongs to the genus Penicillium, 9.54%

belongs to the genus Fusarium and 15.53% Aspergillus genus.

23. Over 144 of samples chemistry, biochemistry and microbiology

analyzed have allowed the development of mathematical correlations among:

strong inverse relationship between starch content and ash (R2 = 0.35, r = 0.39),

the relationship between the starch and oil (R2 = 0.64 and R

2 = 0.76), oil and ash

content (R2 = 0.29, r = 54).

24. Coefficient of determination R2 = 0.82 obtained show that for the

studied hybrids aflatoxin content variation is due to variations in load of fungi of

the genus Aspergillus in a percentage of 82%.

25. Similarly, the levels of ochratoxin was positively correlated to the load

of Penicillium spp fungus (r = 0.87) and zearalenone with the load of Fusarium

spp there is a correlation index R = 0.83.

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