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
Ing. Ioan Aurel Pop Abstract of PHD Thesis
3
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
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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;
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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.
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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.
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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.
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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).
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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%.
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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.
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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.
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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%.
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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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.
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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.
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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.
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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.
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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.
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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.
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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.
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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
Ing. Ioan Aurel Pop Abstract of PHD Thesis
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.