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PhD THESIS

Research regarding the influence of

applying conventional and

unconventional methods for disease

control within local populations of

tomatoes in greenhouse

(SUMMARY OF THE PhD THESIS)

PhD student Nagy (Szabó) Gyöngyi

Scientific coordinator Prof.univ.dr. Carmen Emilia Puia

2016

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Introduction

The increasing demand for organic products is an outstanding economic potential of the tomatoes local varieties, which are the most suitable for growing in ecological systems. The local varieties of tomatoes can generate significant revenues for the rural population and stimulates in this way the preservation of the genetic diversity in farm (ZEVEN, 1998).

The local varieties of tomatoes are very important for the subsistence agriculture and continues to have an important role on the so-called "niche market". Linking the local varieties of tomatoes specific to certain areas and localities can contribute to strengthening the local cultural identity (VETELÄINEN and al., 2009).

The local varieties of tomatoes are characterized by high heterogeneity. They have the advantage of being much better adapted to biotic and abiotic stress conditions (diseases, pests, drought, low nutrient, etc.) and to have excellent taste qualities that could justify a higher selling price than commercial populations of tomatoes (SANCHEZ and al., 2008).

The antioxidant levels in organic tomatoes is higher than that of conventionally grown vegetables, according to a recent study conduct by spaniards scientists. Organic farming is carried out without chemical fertilizers based on nitrogen, therefore the plants grown in this manner activate their own defense mechanism against the pests and this process increases the antioxidant levels (ANNA VALLVERDÚ–QUERALT ET AL., 2011).

Literature review

3. The principal diseases of tomato crop The tomatoes during their development from seed until harvesting

and during storage are affected by various biotic and abiotic factors which may affect their health. The transition from health state to disease state is a process that is achieved gradually and requires an intermediate situation which may be reversible if the factors that caused it disappear. Diseases caused by infectious microorganisms, such as fungi, bacteria, viruses and nematodes, may spread very quickly from one plant to another in favorable environmental conditions (DAMICONE and LYNN BRANDENBERGER, 2010).

Pests and diseases can cause significant damage, reducing the quality and quantity of tomatoes. For the prevention and their treatment are used efficient pesticides. Increasing doses of pesticides in order to obtain reliable results in the control of pests and diseases may cause adverse effects, including the accumulation of large amounts of residues in the obtained products (MANUELA CIOBANU, 2013).

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An alternative approach for the use of pesticides is the integrated control, this includes the use of various methods like biological control of pests and diseases with pheromones and microbial pesticides, genetic engineering, antagonistic insects (MANUELA POGĂCEAN şi MARIA GAVRILESCU, 2009).

Currently, the researches on plant protection of vegetables crops are directed towards finding ways of obtaining maximum crop in terms of conservation and active and dynamic potentiation of unfertilized system exploited. Different soil bacteria and fungi are able to colonize the plant roots and may have beneficial effects on them. These microorganisms can interact and form endofite associations, thus influencing the growth of plants, their yield and their resistance to disease (ROSA HERMOSA et al., 2012).

1.1. The mycoses and diseases produced by the pathogens

similar of fungi In this chapter are listed and described diseases caused by fungi

and pathogens similar to fungi.

1.2. The bacterial diseases of tomatoes Subchapter which presents the bacterial diseases of tomatoes and

the description of the pathogen. 1.3. The virus diseases of tomatoes The content of this subchapter includes the listing and description

of virotic diseases of tomatoes. 4. Control methods for the diseases of tomatoes crop The tomatoes crop from protected areas is very important from the

economic point of view, therefore requires a rigorous control of pests and diseases. Lately, the producers adopted a method of integrated management of pests and diseases, involving various strategies like agro, chemical and biological (SHIPP, 2013). 2.1. Conventional methods The issue of integrated control is particularly complex especially in vegetable crops, which required more than in any other sector of agriculture to combine harmoniously and effective measures to fight against all pathogens in a complete diagram of control also taking into account eliminating or at least reducing the negative side effects of pesticides and obtaining of vegetables with the pollution as low as possible (MARINESCU și colab., 1986).

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The integrated control has the task of protecting the environment and also protecting and maintaining biological balance of species useful in agro-ecosystems (CARMEN PUIA 2003).

2.2. Unconventional methods The alternative methods in plant protection have a high level of

durability. A significant percentage in unconventional methods it has the

biological control. The biological control means the inhibiting of the infection development or reproduction caused by an organism using another organism. The microorganisms used in the biocontrol can act in different ways: by hiperparazitism, antagonism, bacteriophage, as well as the antibiotic, the competition for nutrition and space or by inducing resistance to plants (HEYDARI and PESSARAKLI, 2010).

2.2.1. The use of vesicular arbuscular mycorrhizas in the diseases control from the tomatoes crop

The mykorrhizal fungi are an essential part of the ecosystem, they represent a key component of rhizosphere because they form symbiotic associations with approximately 90% of the plant species from the earth (SCHEFFKNECHT et al., 2006).

The mykorrhizas are the results of an association where the roots of plant are colonized by specific fungi. The plants and mykorrhizal fungi are in a symbiotic relationship or mutual physiologically balanced parasitism called mutualism (IRINA GREBENIŞAN, 2007).

The mykorrhizal fungi supplying the plants with nutrients and water in exchange for some elements assimilated by plants through photosynthesis (KARIN HAGE-AHMED et al., 2013; SREEVANI and REDDY, 2004).

The mykorrhizal fungi forms a physical barrier that prevents access of phytopathogens from the soil. The fungal mantle cover the most fragile parts of roots. Many mykorrhizal fungi cause volatile acids with fungistatic and antifungal effect that reduces the growth of microorganisms, while maintaining a balance between symbiotic fungi and pathogens in the soil. (IRINA GREBENIŞAN 2007).

The arbuscular mycorrhiza and its role of biological protection is of great interest in the context of sustainable agriculture (KARIN HAGE-AHMED et al., 2013).

Considering all these benefits, the mykorrhizal fungi are used like bio-inoculants in agriculture and forestry. The bioproducts are produced by growing endo-mykorrhizal fungi on the roots of plants with high vegetative growth. For example the fungus Glomus is cultivated on the roots of salad to obtain the commercial VAM product (IRINA GREBENIŞAN, 2007).

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2.2.2. The use of bacteria in the disease control from the

tomato crop As use of chemical fungicides in large quantities has not led to the

expected effects it has been required the orientation towards non-chemical sources, namely the bacterial products to combat infectious diseases of crop plants (LUIZA JECU şi colab., 2009).

Promoting the plant growth using rhizobacteria is based on the production of growth regulators, on the plants protection against attacks pathogens and nutrient mobilization (KLOPPER et al., 1999).

The bacteria are the largest and most active group from the soil, being involved in the carbon cycle, nitrogen, phosphorus, sulfur; participating in the mineralization processes ensuring soil fertility and plant nutrition; participates in the solubilizing the organic and inorganic compounds; fix the atmospheric molecular nitrogen needed for the plant nutrition; participates in the formation and degradation of humus (KLOPPER et al., 1999).

Several biofertilizers is based mainly on rhizobacteria, they have positive effects on plant development, mainly related to increased availability of nutrients for plants (VESSAY, 2003).

Personal contribution

3. Research objectives

The present study examines the use of bioproducts with AMF and rhizobacteria in protected crop of tomatoes local populations.

To complete the theme approached it was carried out a comparative study of three local populations of tomato being established the following objectives:

Monitoring the quality of tomatoes seeds selected from the local population.

The study of the impact that application of conventional and unconventional methods have on the development parameters of tomato plants.

The study of the effect that useof the conventional and unconventional methods have on the level of mykorrhizal colonization in the root system of tomato plants.

Monitoring the occurrence of main pathogens in all three populations of tomatoes as a result of treatments and fertilization applied.

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4. The materials used and the applied methodology

4.1. Vegetal material In the experience carried out were used three populations of

tomatoes, selected from the local population: Hostáti, Pink and Ox Heart. The local population Hostáti It's a traditional cultivar with

undetermined growth used both in the field and in greenhouses. The cultivar is vigorous, semi-early, which cultivated in regions of origin is resistant to both, biotic and abiotic stresses and providing a stable and reliable production without the need for use of chemical substances. The fruit of this variety is round-oval, red, with an average weight of 150-200g. The fruit pulp is firm, fleshy with a taste refreshing, sweet and sour, an ideal combination of acidity and sugars. Under optimum environmental conditions form on average four to seven fruits/inflorescence. Suitable for fresh consumption in salads, in soups, sauces and broths.

The local population Pink it is a traditional variety with undetermined growth, used both in field crops and in greenhouses. It is a vigorous and semi–early cultivar. The fruit of the variety is flattened, pink colored, with an average weight between 200-400g. The fruit pulp is fleshy, having a sweet taste. Under optimum conditions of environmental factors the plants produce an average four to six fruits/inflorescence. The fruits does not present a green aura around the stems, the ripening fruit are uniform. Suitable for fresh consumption and processing.

The local population Ox Heart it is a traditional variety with undetermined growth, used both in field crops and in greenhouses. It is a very vigorous cultivar with large heart-shaped fruit. It’s a semi-early variety, for food, with fruit voluminous and smooth. The pulp of the fruit is fleshy, with refreshing sweet taste and a discreet flavor. Grown in areas of origin, they are more resistant to stress than hybrids, both abiotic and biotic stress and provides a stable and reliable production. In favorable environmental conditions the plants produce on average four to seven fruits/inflorescence.

4.2. Bioproducts used The bioproducts used as fertilizer in the experiments were:

o a product based on mykorrhizal fungi, Mykosoil, o a product based on bacterial strains Bactofil

4.3. The methodology applied in the laboratory experiments

4.3.1. The analysis of the pathogenic load of the seed The research method for monitoring the microflora from the seeds

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was the testing on adsorbent paper, analogously to wet chamber (CRISTINA RAICU și DOINA BACIU,1978).

4.3.2. The analysis of root samples to determine the level of mykorrhizal colonization

Staining of samples for achieving that phase it was modified the method described by Hayman and Phillips (1970) and developed by Vierheilig et al. (1998), the modification was accomplished by Stoian (2007).

The estimation of mykorrhizal colonization was performed according to the parameters proposed by Trouvelot et al. (1986, citat de www.dijon.inra.fr):

The frequency of mykorrhiza in the root system (%) The intensity of mykorrhizal colonization in the root system (%) The intensity of mykorrhizal colonization in the root fragments

(%) The arbuscular abundance in mycorrhized parts of root fragments

(%) The arbuscular abundance in the root system (%)

In order to create a more accurate and comprehensive overview of the degree of mykorrhizal colonization was adapted the formula for calculating the degree of attack used in assessing the plant health of the agricultural crops. In this study the degree of attack was called degree of colonization (%).

4.4. The methodology applied in the field experiments To carry out the researche, the experimental field was located in a

greenhouse with the length of 19.2 m and width of 6 m, located in Dâmbul Rotund.

The research was organized as a polifactorial experience and the method of settlement was the latin rectangle, in three repetitions, with the following experimental factors:

The factor 1 Cultivar

o Hostáti o Pink o Ox Heart

The factor 2 The fertilizer

o Unfertilized o Mykosoil o Bactofil

The factor 3 The treatment

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o Conventional (Ridomil, Bravo) o Unconvențional (Bordeaux mixture, macerated

nettle) From the combination of the three experimental factors were

obtained 18 different experimental variants. In each experimental variants were planted six tomato seedlings with a planting distance of 80 cm between rows and 33 cm between plants/row. The unfertilized variant is considered the witness of experience.

5. Results regarding the quality of seeds On the seeds of the three local populations of tomato were

identified Cladosporium fulvum, Alternaria sp. and Aspergillus niger. The highest frequency had the pathogen Cladosporium fulvum on the seed of Ox Heart cultivar, in a percentage of 16.66%. The highest frequency of the pathogen from Alternaria sp. it was reported on the seed of Pink cultivar in proportion of 10%.

6. Results regarding the growth parameters of the tomato

plant Analyzing the parameters of plant development from this three

tomato cultivars studied, during the three experimental years, was achieved by conducting observations and measurements on growth dynamics in different sequences of vegetation, on the level of fructification in different levels of plant development and on the production parameters.

6.1. The analysis of growth dynamics The growth dynamics of plants for all the three cultivars of

tomatoes studied was analyzed in four sequences of vegetation, from 10 to 10 days starting on day 20 after planting, for all the three experimental years (2013, 2014 and 2015).

In the context of the interaction analyzed the cultivar has the greatest impact on the development of tomato plants.

In terms of the analyzed parameter the Hostáti cultivar had recorded the lowest values, followed by Pink cultivar and finally the Ox Heart cultivar with the highest rates of growth dynamics, suggesting the influence more pronounced of the experimental factors on this cultivar.

6.2. The analysis of production parameters In terms of production parameters were made measurements for

the average fruit weight, the average production per plant and the total production per hectare obtained during the all three experimental years.

6.2.1. The analysis of fructification The level of fructification of the plants from the three populations

of tomato was examined on the first three levels of plant development, levels considered more representative, during the all three experimental

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years. The positive influence of the treatment and fertilization applied

during vegetation regarding the fructification of plants it was evident for all the three local tomato populations studied throughout experience.

The highest level of fructification, 73.14% was reached by cultivar Ox Heart in ecological condition and fertilized with Bactofil for the experimental year 2013.

6.2.3. The average weight of the fruits The cultivar with the average weight of fruits most strongly

influenced by the fertilization and treatments applied during vegetation was Pink cultivar. In 2013 this cultivar in terms of conventional treatment and additional mykorrhization recorded the highest fruit of 314,37g.

6.3.2. The average production per plant The average yield obtained per plant under the influence of the

fertilization and treatments applied highlighted the cultivar Ox Heart as having the highest values for the average production per plant, but also the positive effect of the Bactofil application for all three local populations of tomatoes studied.

The additional mykorrhization significantly influenced the average production per plant for the cultivar Ox Heart, this tomato cultivar reached the highest value for 2013 in terms of ecological treatment, 4,68kg / plant.

6.3.3. The total production The beneficial influence of applying additional mykorrhiza and

bacteria can also be observed in the case of total production for all three local populations of tomato studied, regardless by the type of treatment applied.

The cultivar with the highest productivity is the cultivar Ox Heart recording the highest value in 2013 with 179,87t/ha for the variant in terms of ecological treatment and fertilized with mykorrhizal fungi.

The lower production was registered by Pink cultivar in terms of conventional treatment and it has increased under the influence of Bactofil mix, 104,23t / ha in 2014.

7. Results regarding the mycorrhizal colonization of tomatoes plants root system

To estimate the level of mykorrhizal colonization from the root system of tomato plants in the three experimental years were analyzed the effect of treatments and fertilization applied during vegetation period on the intensity of cortical colonization and the percentage of arbuscular colonization.

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7.1. The analysis of cortical colonization intensity The cortical colonization intensity was measured and analyzed

during the three experimental years in three sequences of vegetation. The mykorrhization of root systems is dependent more on

fertilizer and less by the cultivar studied. Although the frequency of mykorrhization is 100%, the intensity of

colonization is less than 15%. The positive influence of additional mykorrhization can be seen throughout experiences, the most influenced cultivar was the Ox Heart that recorded in 2015 the highest value 11.90% under ecological treatment and additional mykorrhization.

7.2. The analysis of arbuscular colonization percentage To estimate the percentage of arbuscular colonization was analyzed the influence of experimental factors, the fertilization and treatments applied during the growing season on arbuscular abundance in mycorrhized parts of root fragments as well on arbuscular abundance of whole root system of all the three tomato local populations studied.

7.2.1. The arbuscular abundance from the mycorrhized parts of the root fragments

The analysis of the arbuscular abundance from the mycorrhized parts of the root fragments was performed for each experimental year within three vegetation sequences per season,20, 40 and 60 days after planting.

The positive influence of additional mykorrhization on arbuscular abundance from the mycorrhized parts of the root fragments was notice on all three local populations of tomatoes.

The analyzed parameter reaches its highest value in 2014 for the variety Hostáti in conventional treatment and additional mycorrhization, 64.24%.

7.2.2. The arbuscular abundance of root system Arbuscular abundance in the root system was significantly affected as a result of fertilization with mycorrhizae and the maximum value was recorded in 2013 for the cultivar Ox Heart under conventional treatment, 5.18%.

8. Results on the incidence of the main pathogens in the experiments

The only disease occurs in experience was early blight produced by the pathogen Alternaria solani. Within the tomato cultivar Ox Heart no attack of the pathogen Alternaria solani was manifested in 2013.

The attack was not very strong neither for the other two tomato populations, which is why attack degree showed no significant differences from control

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The highest attack degree was observed in the cultivar Pink in 2014 to 4.17% for the variant without additional fertilization and treated conventionally. 9. Conclusions and recommendations 9.1. Conclusions concerning the quality monitoring of tomato seed selected from the local population

9.1.1. On the seeds of tomato local populations studied were identified Cladosporium fulvum, Alternaria sp și Aspergillus niger.

9.1.2. The Cladosporium fulvum pathogen had the highest frequency on the seed of Ox Heart in a proportion of 16.66%.

9.1.3. The highest frequency of Alternaria sp pathogen was reported on the seeds of Pink cultivar in a proportion of 10%.

9.2. Conclusions concerning the study of the impact resulting

from the application of conventional and unconventional methods on the development parameters of tomato plants

9.2.1. The influence of mykorrhizal and bacterial mixture on plant growth is evident during the first sequence of vegetation up to 50 days from planting, at which point the differences between the plants were not significant.

9.2.2. The level of plant fructification from the three local populations of tomato was significantly influenced on the first three levels of development as a result of additional fertilization with mykorrhizae and bacteriae.

9.2.3. The additional fertilization with bacterial and mykorrhizal mixture positively influenced the development of tomato plants, both in terms of growth and production parameters. This contributes to achieving a greater income economically.

9.3. Conclusions concerning the study of the effect that it has

the use of conventional and unconventional methods on the level of mykorrhizal colonization of the root system of tomato plants

9.3.1. Differences in conditions of additional mykorrhization are visible throughout the monitoring period, additional mykorrhization acts like a starter in the first two sequences of vegetation and maintain, with slight fluctuations, the arbuscular percentage in mycorrhized fragments at a level of about 50% throughout the period of experimentation.

9.3.2. In terms of additional mykorrhization the tomato plants predominantly accessing these resources to the detriment of existing soil microflora.

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9.4. Conclusions concerning the monitoring of the occurrence of the main pathogens in the three populations of tomatoes due to fertilization and treatments achieved

9.4.1. Using bioproducts improved the tolerance of tomato plants to various pathogens attack, the only disease occurs in experience was early blight produced by the pathogen Alternaria solani.

9.4.2. Applying additional fertilization with mycorrhizae and bacterial bioproduct has positive effects on reducing the attack of the pathogen Alternaria solani for all three populations studied under the influence of both conventional and organic treatments.

9.5. Recommendations 9.5.1. It is recommended to spread these three local populations of

tomato in protected crops, due to productivity rather high and adaptability to the treatments and fertilization applied to vegetation.

9.5.2. It is recommended to use bioproducts in tomato crop because they have positively influenced on the parameters of growth and production contributing to a greater economically income.

10. Innovative contributions of the thesis 10.1. The three local populations of tomato Hostáti, Roz și Ox Heart was taken for the first time in a scientific study.

10.2. This study analyzes the potential of using the vesicular arbuscular mykorhizae associations (Mykosoil) and rhizobacteria (Bactofil) in protected crop of tomatoes local populations tomato.

10.3. The measurement of the relationship between mykorrhizal and rhizobacterian inoculum is carried out under conventional and unconventional condition of crop, in order to assess the applicability and adaptation to local conditions of exogenous microflora products.

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