Unravel the beneficial effects induced by Trichoderma harzianum on tomato plants: the intimate dialogue that improves growth and defense responses.

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Unravel the beneficial effects induced by

harzianum on tomato plants: the intimate dialogue that

improves growth and

Agriculture, Food and Environment

Department of Agriculture, Food and Environment

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Unravel the beneficial effects induced by Trichoderma

on tomato plants: the intimate dialogue that

improves growth and defense responses.

by

Lisa Fiorini

Ph. D. Thesis

Agriculture, Food and Environment

Department of Agriculture, Food and Environment

University of Pisa

Trichoderma

on tomato plants: the intimate dialogue that

responses.

Department of Agriculture, Food and Environment

University of Pisa

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Unravel the beneficial effects induced by

tomato plants: the intimate dialogue that improves growth and

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Agriculture, Food and Environment

Candidate: Lisa Fiorini

Supervisor(s)

Prof. Name Surname Giovanni Vannacci

Accepted by the Ph.D School

The Coordinator

(Prof. Name Surname) Alberto Pardossi

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Unravel the beneficial effects induced by Trichoderma harzianum

tomato plants: the intimate dialogue that improves growth and

defense responses.

by

Lisa Fiorini

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy in

Agriculture, Food and Environment

Giovanni Vannacci

D School

Name Surname) Alberto Pardossi

Date: ____________

Trichoderma harzianum on

tomato plants: the intimate dialogue that improves growth and

A thesis submitted in conformity with the requirements

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Declaration

This thesis is a presentation of my original research work. Wherever contributions of others are involved, every effort is made to indicate this clearly with due reference to the literature and acknowledgement of collaborative research and discussions.

This thesis contains no material that has been submitted previously, in whole or in part, for the award of any other academic degree or diploma.

Questa tesi è il risultato di un mio lavoro di ricerca originale. L’eventuale contributo di altri a questa tesi è stato adeguatamente indicato attraverso le citazioni bibliografiche e il riconoscimento degli studi condotti in collaborazione.

Questa tesi non contiene materiale che è stato presentato in precedenza, in tutto o in parte, per il conferimento di qualsiasi altro titolo o diploma accademico.

Signature/firma Date/data

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Preface

This thesis interpolates material from two papers by the author (Baroncelli et al. 2015; Fiorini et al. 2015). Chapter 2 uses material from References Fiorini et al., (2015), co-authored with other authors from Pisa University and CNR of Monterotondo Scalo, Rome. Some material from paper

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Abstract

The increased need for food availability combined with the request of a more sustainable way of production, lead researchers to find alternative to chemical pesticides and fertilizers. Many Trichoderma isolates are already available as active ingredients of commercial biopesticides. Their use would reduce chemical inputs, in a perspective of sustainable agriculture, allowing natural resource conservation. The potential of Trichoderma harzianum T6776 (T6776) to be used as a biocontrol agent and as a biostimulant has been previously proven on several cultivars of tomato plants. In order to investigate the mechanisms behind these effects, the genome of T6776 was firstly sequenced and a phylogenetic analysis was performed, thus allowing to correctly identify our strain as a T. afroharzianum, a new species included into the T. harzianum species complex. Several biochemical analyses were done in order to elucidate the mechanisms responsible for growth promotion of the tomato cv. Micro Tom, and these analyses highlighted the complex metabolic change induced by T6776 in tomato. Growth promotion resulted to be a consequence of a changed sugar allocation, that influences the photosynthetic efficiency of the plants, combined with an increased level of growth related hormones, such as indoleacetic acid. Moreover, in preliminary tests, we showed that T6776 is able to increase plant tolerance to biotic and abiotic stresses. Plants grown in presence of T6776 responded better to pathogens attack, such as Fusarium oxysporum f. sp. radicis lycopersici and Alternaria solani. Likewise, the photosynthetic efficiency of salt stressed plants treated with T6776 increased, compared with untreated plants, suggesting an enhanced salt tolerance induced by T6776. The increased level of the stress related hormone jasmonic acid (JA) found in roots and xylem vessel of T6776 treated plants could in part explain the increased plants tolerance against biotic and abiotic stress. In addition, the high level of starch found in roots of treated plants could help plants to face to abiotic stress, representing an extra energy reserve. In order to identify molecules involved in root colonization process by T6776, a specific class of hypothetic effector proteins were identified and characterized in the T6776 genome. The expression levels of nine LysM encoding genes identified in the analysis were measured using qPCR in T6776 interacting with tomato roots. Analysis revealed that the expression levels of 4 LysM encoding genes increased during the early stage of the interaction between T6776 and tomato plants. These results show, for the

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first time, the possible involvement of LysM effectors in the interaction between a Trichoderma sp. isolate and the host plants.

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Acknowledgments

Now, the writing of my thesis has come to the end, and I am here thinking about how many people I am grateful. Last three years were not easy, and it was a long journey with a lot of up and down. I would like to thanks every person which help was fundamental for achieve this result.

Above all, I would thank my family, particularly my mother and my brothers and sisters, that during this journey they always believed and supported me, with patience and love, especially when I didn't.

This work would have not be completed without support, supervision, guidance and encouragement of many people who have contributed during last three years.

Firstly, I would thank my Tutor Prof. Giovanni Vannacci, for his patience and availability for discussion, which enabled my thoughts to mature and grow. My sincere gratitude goes also to Sabrina Sarrocco and at all the staff of the Laboratory of Micology and Phytopathology of Pisa University, to Maria Rosaria Vergara, to Susanna Pecchia to Grazia Puntoni and Antonio Zapparata, but also to many students that passed through our laboratory during their thesis, particularly to Stefania Diquattro, Luigi De Martino, Giulia Beatrice Piaggeschi, Federico Rossi and Giulia Vecchiotti.

I would like to remember here Maurizio Forti, technician of our laboratory, who passed away in 2013, that helped me a lot with his advices about science and life.

I am grateful to Professor Bart H.J. Thomma of the Wageningen Research Centre that gave to me the chance to work upon his laboratory with his group, the Verticillium group. My sincere gratitude goes particularly to Mireille Van Damme, David Cook and Eduardo Rojas Espadillas. Their expertise in molecular biology and their great patience in helping me were fundamental for a part of this work.

I would like to thanks also Piero Picciarelli, Lorenzo Guglielminetti, Maurizio Curadi and Lorenzo Mariotti from Pisa University and Andrea Scartazza from CNR of Monterotondo Scalo, from which collaboration we published a very interesting paper.

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A special thanks goes also to the Prof. Mario Enrico Pè of the Sant'Anna University of Pisa and to the Prof. Alberto Pardossi of Pisa University. I would thank Enrique Monte of the Salamanca University and Irina S. Druzhinina of Vienna University of Technology (TU Wien) for having accepted to review this thesis.

A very lovely thanks goes to my aunt Sandra Sheller, writer and head of the English Department of Rider High School (Texas; USA), for English review of this thesis.

At the end I want to thanks Riccardo Baroncelli, which helped me in several bioinformatics analysis and for everything he teach to me.

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List of Tables

CHAPTER 2:

Table 2.1: Strains of T. harzianum species complex used in the phylogenetic analysis for the identification of the T6776 species. In the table are reported the geographic origin, the substrate from which the strain was isolated and the accession number for each genes.

CHAPTER 3

Table 3.1: Tomato hydroponic solution (THS) chemical composition used in the hydroponic system.

Table 3.2: Chemical composition of Hoagland solution used in the soil system.

Table 3.3. Photosynthetic parameters of not inoculated (CNT) and inoculated with T6776 (T6776) tomato plants, 5 weeks after planting (28 DPI) in hydroponic condition. Values represent the Mean±SE of 8 replicates. At different letters within the same column correspond values significantly different, according to Tukey's test (P<0.05). CNT: control plants; T6776: plants inoculated with T6776; PPFD: Photosyntetic photon flux density. a Maximum photochemical efficiency of Photosystem II (PSII); b Actual photochemical efficiency of PSII; c

Non photochemical quenching; d Net CO2 assimilation rate; e Stomatal conductance; f Internal

CO2 concentration.

Table 3.4: Hormonal profiling (JA, SA and IAA) of root and xylem exudates of 5 week old (28DPI) plants inoculated (T6776 and T6776(s)) or not inoculated (CNT and CNT(s)) with T6776 determined by GC-MS. Concentrations are expressed in ng g -1 fresh weight (FW) for root samples and in mM for xylem exudates. Values are means ± SE (n=6 and n=3 in hydroponic and soil, respectively). Data were submitted to one-way ANOVA and significant comparisons between treatments, performed by Tukey’s test, are marked with different letters.

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List of Figures

CHAPTER 2:

Figure 2.1: Bayesian MCMC analysis tree constructed from the alignment based on the concatenation of act, cal, its, rpb2 and tef1 partial sequences of 29 Trichoderma harzianum species complex isolates. The tree was rooted with sequences from T. virens. Only branches with a posterior probability of at least 0.95.

CHAPTER 3

Figure 3.1: Increase in height of inoculated (T6776 and T6776(s)) and non inoculated (CNT and CNT(s)) plants by T6776. Each point represents mean + SD of 3 replicates, 12 plants each

(n=36), from the 10th_-15th_{day post sowing (DPS) to 35}th _{DPS (from 5 to 28 days post}

inoculum-DPI). a: hydroponic condition; b: soil condition.

Figure 3.2: Leaf area parameter of plants grown in soil condition not inoculated (CNT(s)) and inoculated (T6776(s)) with T6776. Each point represent the MEAN±SD of 12 plants each, per replicates (n=36).

Figure 3.3: Biomass quantification of plants grown in hydroponic (a) and soil (b) condition. Fresh and dry weight of leaves, stems, roots and whole weight of not inoculated (CNT and CNT(s)) and inoculated with T6776 (T6776 and T6776(s)) tomato plants, 5 weeks after planting (28 DPI). a: hydroponic condition; b: soil condition. Values represent the mean±SE of 3 replicates of 5 plants each (n=3). At different letters within the same organ correspond values significantly different according to Tukey’s test (P<0.05). CNT and CNT(s): not inoculated plants grown respectively in hydroponic and soil; T6776 and T6776(s): inoculated plants grown respectively in hydroponic and soil.

Figure 3.4a: Photochemical efficiency of Photosystem II (PSII) of T.6776 treated (T6776) and

not treated plants (CNT), determined at light growing condition intensity (Photosynthetic Photon Flux Density (PPFD) of 200mmol m-2_s-1_{) and at higher light intensity (300 mmol m}-2_s-1_).

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treatments (n=36), determined at 14, 21 and 27 days post T6776 inoculum (DPI). Meaningful differences (p< 0.05) between treatments revealed by ANOVA analysis were marked with different letter.

Figure 3.4b: Photochemical efficiency of Photosystem II (PSII) of T6776 treated (T6776(s))

and not treated plants (CNT(s)), determined at light growing condition intensity (Photosynthetic

Photon Flux Density (PPFD) of 200mmol m-2_s-1_{) and at higher light intensity of 300 mmol m}-2_s

-1 _{. Values represent Means ± SE of 6 plants grown in soil condition per 3 replication per}

treatments (n=18). Meaningful differences (p< 0.05) between treatments revealed by ANOVA analysis were marked with different letter.

Figure 3.5: Light response curves of photosynthetic CO2 assimilation rate (A) (left panel) and relationships between A and intercellular CO2 concentration (Ci) at different light intensity

(PPFD) (right panel) in 5-weeks-old (28 DPI) inoculated (T6776) and not inoculated plants (CNT) grown in hydroponic condition. Values are means ± SE (n=3).

Figure 3.6: Chlorophyll (a, b, total) and carotenoids contents of fully expanded leaves in 5-weeks-old (28 DPI) not inoculated (CNT and CNT(s)) and inoculated with isolate T6776 (T6776 and T6776(s)) tomato plants. a: hydroponic condition; b: soil condition.. Values represent the mean ± SE of 9 replicates for treatment (3 samples for each replicates, 3 replicates for treatment, n=9). At different letters within the same column correspond values significantly different, according to Tukey's test (P<0.05). CNT: control plants in hydroponic; T6776: plants inoculated with isolate T6776 in hydroponic, CNT(s): control plans in soil; T6776(s): plants inoculated with T6776 in soil.

Figure 3.7: Carbohydrate content at leaves, stems and roots level of 5-weeks-old (28 DPI) not inoculated (CNT and CNT(s)) and inoculated with T6776 (T6776 and T6776(s)) tomato plants. a: hydroponic condition; b: soil condition. The content of carbohydrates was expressed in µmol of hexose equivalent for 1 gram of fresh weight (FW) of leaves, stem and roots tissue. Values (µmol hexose equiv g-1 FW) represent the mean±SE of 3 replicates for treatment (n=3). At different letters correspond values significantly different, according to Tukey's test (P<0.05). a:

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CNT: control plants in hydroponic; T6776: plants inoculated with T6776 in hydroponic. b:

CNT(s): control plants in soil; T6776(s): plants inoculated with T6776 in soil.

CHAPTER 4

Figure 4.1: Percentage of emerged plants were recorded seventeen days after sowing and inoculation with pathogens and/or T6776 in order to evaluate the effect of the co-inoculation of T6776 with pathogen on plants emergency. Percentage data were subjected to angular transformation before statistical analysis to normalize the distribution and stabilize the residual variance. All the data were analysed using one-way ANOVA. Pairwise comparisons were performed using the Tukey test by SYSTAT 1.2 assuming P<0.05 as the significance level and significant differences between treatments were marked with different letters. CNT: untreated plants; T6776: plants treated with T6776; FORL: plants treated with F. oxysporum f.sp. radicis lycopersici; T6776+FORL: plants treated with F. oxysporum f.sp. radicis lycopersici and T6776; RS: plants treated with R. solani; T6776+RS: plants treated with R. solani and T6776; MP: plants treated with M. phaseolina; T6776+MP: plants treated with M. phaseolina and T6776. Figure 4.2: Area and diameter of the lesion produced by the air borne pathogen Alternaria solani on tomato leaves of 5 week old, treated at sowing time without (AS) or with T6776 (T6776+AS). The total area of the leaflet, the lesion area and the lesion diameter were measured

using ImageJ and expressed in cm2_{and cm. Values represent Mean ±SE of 24 measurements per}

3 replications per treatments (n=72). The data were analysed using one-way ANOVA. Pairwise comparisons were performed using the Tukey test by SYSTAT 1.2 assuming P<0.05 as the significance level and significant differences between treatments were marked with different letters.

Figure 4.3: Actual efficiency of PSII photochemistry in the light (PSII) of inoculated (T6776salt)

and not inoculated (CNTsalt) plants, at growing light (PPFD 200µmol m-2 s-1) and after 30

minutes of acclimation at 300µmol m-2_s-1 _{of PPFD. Measurements were taken at time of stress}

imposition (0) and after 24 and 96 hours after stress. Values represent the Means ±SD of 5 replicates. Data were submitted to One way ANOVA and significant comparison between treatments, performed by Tukey's test, are marked with *(*P<0.05; **P<0.01; ***P<0.001).

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Figure 4.4: Actual efficiency of PSII photochemistry in light (PSII) of inoculated (T6776anoxia)

and not inoculated (CNTanoxia) plants in the dark, at growing light (PPFD 200µmol m-2 s-1) and

after 30 minutes of acclimation at 300µmol m-2_s-1 _{of PPFD. Measurements were taken after 60}

minutes of air recovery from eighteen hours of anoxia stress imposition. Values represent Means±SD of five replicates for treatment. Data was submitted to one-way ANOVA and significant differences between treatment, performed with Tukey’s test, were marked with * (*P<0.05; **P<0.01; *** P<0.001).

CHAPTER 5

Figure 5.1: Modular structure with conserved domain of nine identified LysM encoding genes of isolate T6776. The domain identification was performed using different database and marked by different colours: blue: SMART, light purple: Pfam, green: Phobius; purple: ProDom.

Figure 5.2: Domain structure of predicted proteins encoding genes belong to the hypothetical cluster 1 (scaffold 0028) and cluster 2 (scaffold 0137). The two hypothetic cluster present one gene each encoding for a SgC chitinases that present similar structure with the exception of the Hce2 domain found in Th_05014 and not in Th_01572; one genes each encoding for a LysM protein with 3 or 4 LysM motifs (Th_01574 and Th05014, respectively) and one gene each encoding for an unknown secreted protein (Th_01573 and Th01513).

Figure 5.3: Expression profile of 9 LysM encoding genes identified in the T6776 genome sequence. Data are Means±SD of three biological replicates, collected 24 hours post T6776 inoculation. T6776: T6776 grown in MS; T6776+MT: T6776 grown in MS in presence of Micro Tom plants. Th_CAL: T6776 clamodulin genes used as housekeeping genes. Statistic analysis was performed using the SPSS program and significant differences between treatment were marked with different letters (P≤0.05).

Figure 5.4: Expression profile of genes belonging to the cluster 1 and cluster 2. Th_01573 and Th_05013 are not LysM encoding genes. Data are Means±SD of 3 biological replicates, collected 24 hours post T6776 inoculation. T6776: T6776 grown in MS; T6776+MT: T6776 grown in MS in presence of Micro Tom plants. Th_CAL: T6776 clamodulin genes used as

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housekeeping genes. Statistic analysis was performed using the SPSS program and significant differences between treatment were marked with different letters (P≤0.05).

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CHAPTER 1: General introduction and thesis outline

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Chapter 1 GENERAL INTRODUCTION AND THESIS OUTLINE

1. General Introduction

1.1 Importance of biopesticides

From the second half of the last century, crop production significantly increased to meet the needs of a growing human population, but more efforts will have to be taken in the future. One way to increase food availability is to reduce crop losses due to pests. Around 67,000 different crop pest species have been estimated, including plant pathogens, weeds, invertebrates and some vertebrate species, and all together they are responsible for about the 40 per cent of reduction in the world's crops yield (Chandler et al., 2011). Since the 1960s, in the industrialized countries pest management has been based on massive increase in the use of synthetic pesticides (15-20 time), but this approach is not now considered sustainable (Oerke et al., 2006). Chemical control of pests combined with advances in crop techniques have helped to increase crop yields by 70 per cent in Europe and 100 per cent in the United States (Pretty et al., 2008). This wide spread use of chemical pesticides, does not only affect the farmers income, but it impacts all the society at large, compromising human health and poisoning natural environments (Pimentel D., 2005). Such externalities are paid by all citizens. Nowadays, social opinion and government regulations push farmers to find alternatives to chemicals for a more sustainable and secure agriculture (Glare et al., 2012). Integrated pest management (IPM) is a sustainable way to increase food availability and crops yields, by combining different crop protection practices with careful monitoring of pests and their natural enemies (Bajwa et al., 2002; Flint et al., 1981). The IPM system was promoted by many experts and has been centrally placed within the European directive on sustainable use of pesticides (European Parliament, 2009). IPM includes biological control with natural enemies, such as parasites, predatory insect and microbial antagonist of plant pathogens (Glare et al., 2012; Chandler et al., 2010). Just to give an idea of the importance, IPM based on biological control is used, in Europe only, on over 90 per cent of greenhouse tomato, cucumber and sweet pepper production in The Netherlands and it represents a standard practice for greenhouse crops in United Kingdom (Chandler et al., 2011). In Italy

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the area under biological control increased from just 5.6 million ha in 2005 to around 9.6 million ha in 2011. The use of biological control requires considerable knowledge, but it has clear benefits in terms of reliable pest control, lack of phytotoxicity and pathogenicity to humans, to other crops (mushrooms) or other agents of biological control such as entomopathogenic fungi, a short harvest interval after treatments and better crop quality. Biopesticides are a group of plant protection products used in the IPM, which have become an important component of environmentally friendly pest management. They have a range of attractive properties: most of them are selective, produce little or no toxic residues, and with development costs significantly lower than those of chemical pesticides. Disadvantages include a slower rate of kill compared with conventional chemical pesticides, shorter persistence in the environment and susceptibility to unfavourable environmental conditions (Glare et al., 2012). Biopesticides comprise preparations containing living microorganisms, biochemicals, and semiochemicals (pheromones). Within microbial biopesticides, Trichoderma-based products are the most commonly used, and some examples of commercialized ones are Vinevax (Agrimm Technologies, New Zealand), Remedier (Isagro Italia, Italy) and Tusal (Certis Europe, Italy). The biopesticides global market is predicted to grow rapidly in the future (Chandler et al., 2011; Glare et al., 2012), thus, an increase in understanding of the biology and ecology of their active organisms will contribute to the establishment of biopesticides in mainstream agriculture. For example, the occurrence of endophytic microbes with antagonistic abilities is leading to exciting new opportunities because it overcomes delivery issues often associated with biopesticides. Endophytic biocontrol agents can be easily applied to seeds, tissues culture plantlets and in other propagating material, providing protection to biotic and abiotic adversities. Endophytes can also have additional beneficial properties, such as accelerating seedling emergence or promoting plant growth (Compant et al., 2010; Harman GE, 2011). Trichoderma species have been found growing internally within the roots of vegetable, fruit and forest tree crops (Bailey et al., 2008; Harman GE, 2011). Several commercially available products have already been developed to exploit endophytic abilities of pesticidal microbes. One version of Vinevax is a mixture of endophytic strains of Trichoderma harzianum formulated as a dowel that is used in grapevines cultivation which enabled internal colonization of tissues and suppression of a range of woody trunk diseases caused by fungi such as Botryosphaeria and Phaeomoniella (http://agrimm.co.nz/products/vinevax-pruning-wound-dressing/).

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Recently evidences suggest that endophytic microorganisms, which provide protection against diseases, can also provide drought tolerance in certain crops (Brotman et al., 2013; Hubbard et al., 2014; Kim et al., 2012). Additional benefits to pest control can be an important selection criterion in screening programs in order to choose commercially attractive biocontrol isolates and open a significant opportunity for marketing biopesticides as products with an added value (Kohl et al., 2011).

More emphasis on research on these microorganisms will lead to an improvement in biopesticides formulation and to a better understanding of their modes of action. Whole or partial genome sequencing of beneficial microorganisms will be a useful tool to select good isolates with a known mode of action, such as the production of antibiotics or of novel interesting and useful traits.

1.2. Trichoderma harzianum T6776

The process that brings the discovery of a new biocontrol agent has been paved by an elevated number of well designed biological assays required to discover new useful traits. Discovery of new isolates is a good way to ameliorate the efficiency of biopesticides in agriculture, since local strains could be more efficient or adapted to a specific area of cultivation or on specific plant species. Moreover, the availability of new isolates able to induce beneficial effects on plants could be a good opportunity to increase the knowledge about biocontrol agents allowing the comparison among different species and or strains. Trichoderma harzianum 6776 (T6776) was isolated from a soil in Pisa (Italy), and its effects on plants and against pathogens has been studied for long time (Baroncelli et al., 2015; Brondi D. BCs Thesis, 2008; Fiorini et al., 2015; Radicchi G BCs Thesis, 2006; Sarrocco et al., 2013; Tarquini G. MSc Thesis 2014; Vecchiotti G. BCs Thesis; Viva A. MSc Thesis 2003). Several studies have been done on this isolate, it has been tested for plants growth promotion, defence induction in plants, plants root colonization abilities and mycoparasitism against pathogens. Recently, the whole genome sequence of T6776 was released (Baroncelli et al., 2015) and its genetic potential has been explored. T6776 was tested for the first time on tomato (Solanum lycopersicum L.), grown in greenhouse conditions during winter time within a screening experiment for growth promotion and defence induction,. Among the 61 isolates of Trichoderma spp. evaluated for their beneficial effects, T6776 resulted to be a

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good resistance inducer against Botrytis cinerea in vivo, reducing the number of infected plants. Moreover, in dual confrontation assays, growth of the plant pathogen was inhibited before contact with T6776. These results suggest a possible production of antibiotics or diffusible compounds by T6776, and/or a competition for nutrient and space as mechanisms at the basis of B. cinerea growth inhibition in vitro (Viva A., MSc Thesis). T6776 was also tested for root colonizing abilities and growth promotion on fifteen different plant species, such as pepper (Capsicum annuum L.), mint (Mentha L.) and tobacco (Nicotiana tabacum L.) (Radicchi G., BSc Thesis), where T6776 resulted to be a good root colonizer and growth promoter, mainly on tomato and pepper. Semi- quantitative assays were used to evaluate T6776 production of plant cell wall degrading enzymes (CWDEs), such as cellulases and proteases. A screening among 8 different isolates for CWDEs production showed that T6776 is one of the best producers of cellulases, polygalatturonases and proteases, supporting the root endophytic abilities of this strain (Radicchi G., BSc Thesis). Interestingly, a different level of root colonization among the plants species tested was found: T6776 was able to colonize extensively root apparatus of tomato (Solanum lycopersicum L. cv Cuore di Bue), basil (Ocymum basilicum L: cv Foglia di Lattuga), pepper (Capsicum annum L. cv d'Asti rosso) and radish (Raphanus sativus L. cv Capuccetto rosso), whereas a low level of colonization was found in root apparatus of tobacco (Nicotiana tabacum L. cv SR1) and carrot (Daucus carota L. cv Berlicum), and no colonization was found on mint (Mentha L. cv Crispa perenne). These results are an example of the fact that the endophytic abilities depend not only from the strain, but also from the host species. Growth promotion ability on different tomato cultivars and on radish was recently proven, and the biocontrol ability against soil-borne pathogens has been shown. T6776 was found to enhance defense of tomato and radish plants against Rhizoctonia solani, Fusarium oxysporum f. sp. lycopersici and Fusarium oxysporum f. sp. radicis-lycopersici (Sarrocco et al. 2013). To understand the genetic potential encoded by T. harzianum T6776 and to facilitate its use as a model beneficial organism to study plant growth promotion, induced systemic resistance and mycoparasitism, T6776 genome was sequenced and released (Baroncelli et al. 2015). The genome was sequenced using Illumina MiSeq 250bp paired-end reads sequencing technology (Quebec Innovation Centre, Canada). The whole genome sequence was assembled (VELVET 1.2.08) and resulted in 1573 scaffold, total length of 39.73 Mb and GC content of 48.50%. . The T6776 proteome was identified, resulting in 11,501 protein-encoding genes models. 1,412 proteins (12.28% of the proteome) were predicted to be secreted and represent the T6776

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secretome. We compared the T6776 genome sequence with other published Trichoderma genomes (T. reseei, T. virens and T. atroviride) and with genomes of fungi with different lifestyle, such as biotroph (Claviceps purpurea), emibiotroph (Mycosphaerella oryzae and Colletotrichum fiorinae), saprotroph (Neurospora crassa), necrotroph ( Fusarium graminearum). The Clonostachys rosea genome was included in the genome comparative analysis, representing a biocontrol agent belong to another genera. . Among the hypothetical secreted proteins, 63 have been found to be Trichoderma specific and 35 have been found to be T6776 strain specific (Baroncelli et al. 2015). The expansions in gene families were evaluated in the genome of T. harzianum 6776 and compared to other organisms included in the analysis, showing a potential reservoir of enzymes and proteins involved in mycoparasitism and in plant interaction. The major expansions were found in the carbohydrate active enzymes (CaZY), peptidases and proteins involved in the secondary metabolism pathways (Piaggeschi B., MSc Thesis). Among CaZY’s enzymes, glycoside hydrolases (GH5, GH30 GH13, GH2, GH20 GH2/20), chitosanase GH75 and the polysaccharide lyase (PL7) resulted to be expanded. Among peptidases, the aspartic proteases, G1 peptidases and serine peptidases S53 were found to be expanded. Aspartic peptidases have been shown to have a role in mycoparasitism or in inducing defense and stimulating plant growth following Trichoderma-plant interaction (Hermosa et al. 2012). Some proteins identified in this analysis are involved in the polyketide or peptaibols production, products of the secondary metabolism. Usually these enzymes are not secreted out of the cell, since they are used in metabolic pathways Noteworthy, in the T6776 genome some of them were predicted to be secreted, such as some polyketide synthases (INTERPRO family number identification: IPR019587, IPR020842, IPR020843), suggesting their possible interaction with plants or other fungi metabolism. Moreover, proteins belong to unclassified families resulted from analysis, such as epoxide hydrolases (IPR016292) and hydrophobin like proteins.

The previous experimental information combined with the genome availability makes the strain T6776 suitable to be used in plant-Trichoderma and in plant pathogen-Trichoderma interaction studies.

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1.3 Tomato as a model system for plant-microbe interaction studies

Tomato (Solanum lycopersicum L.) is one of the most important crop in the world, with a global production of about 160 million tons of fresh fruits whereof 15 million tons only in Europe (FAOSTAT Database, 2012). Tomato is an alternative model plant to Arabidopsis thaliana due to its diverse developmental traits, such as photoperiod-independent sympodial flowering, formation of fleshy climacteric fruits, composite leaves and multicellular /glandular trichomes. It is also characterized by agronomically important plant-insect and plant-pathogen interactions not found in Arabidopsis (Carvalho et al. 2011). Recently the Tomato Genome Consortium (TGC, solgenomics.net), a multi-national team of scientists from 14 countries, released the annotated genome (TGC, 2012) making tomato a convenient model plant species also for functional genetics and physiological studies. Moreover, its genome resulted highly syntenic with those from other Solenaceae (TGC 2012).

Originally bred with home gardening purpose (Scott & Harbaugh 1989), the laboratory-dwarf tomato cultivar Micro-Tom has been proposed as a model system for genetic (Meissner et al. 1997), hormonal, physiological (Campos et al. 2010) and plant-pathogen interaction studies (Arie et al. 2007). It is characterized by unique features, such as high density growth capacity (up to 1357/m2_{), short life cycle (70-90 days), a high number of available mutants and an}

efficient Agrobacterium-mediated transformation protocol (Sun et al. 2006). Furthermore, this miniature cultivar differs from standard tomato for only two major genes (Meissner et al. 1997). A Japanese Solanaceae Genomic Project (J-SOL) consortium, established with the purpose of sharing genomic resources for tomato, released macroarray profiling data, mutants generated using EMS and full cDNA sequences of Micro-Tom (Saito et al. 2011).

2. Thesis outline

Beneficial effects induced in plants such as growth promotion and enhanced tolerance against biotic and abiotic stress by beneficial isolate are different and their induction is dependent not only by isolate-specific abilities but also by the host plant. Although general mechanism involved in the beneficial effects can be obtained by several Trichoderma spp., variation in specific mechanisms can occur, since they are species and strains specific, such as IAA

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production involved in growth stimulus. Moreover, mechanisms behind the beneficial effects are plural and in some cases synergic, and they are also dependent on the plant species. The main scope of this research work was to investigate the mechanisms behind enhanced growth and stress tolerance induced by T6776 on a model plant system. For this purpose, we set up a system using T6776 and tomato plants cv. Micro Tom, which was used to evaluate plant responses to different conditions. Micro Tom tomato cultivar was chosen for its suitable characteristic, that render it a suitable model plant to be used in laboratory conditions. The genome of T6776 was sequenced and firstly its species characterization is presented in Chapter 2. In order to identify the species of the isolates we worked with, we performed a phylogenetic analysis using 5 unlinked genes sequence (act, cal, its, rpb2, tef1) on 29 isolates belong to the T. harzianum species complex. The analysis took advantages of the T6776 genome availability, rendering quickly the access to gene sequences. Our analysis shows that the isolate T6776 belongs to T. afroharzianum species, and not to T. harzianum sensu strictu. The isolates belonging to the T. afroharzianum species have often been isolated from soil and have tropical origins. In order to investigate the mechanisms behind growth promotion, we evaluated plant responses towards T6776 inoculation, measuring several growth and metabolic parameters on tomato plants and our results are presented in Chapter 3. We used the system T6776- Micro Tom plants, inoculating plants with T6776 using two different formulations and growing tomato plants in two cultivation systems. We observed that T6776 positively affects growth of plants, interfering with hormones levels and sugar partitioning. The photosynthetic rate of plants was improved by the T6776 interaction, as well the pigment content of the leaves. A mechanism of tomato growth stimulation by T6776 is presented and discussed. Part of the results presented in Chapter 3 were recently published in a paper (Fiorini et al. 2015). In Chapter 4 we evaluate the effect of T6776 in ameliorating plant responses against biotic and abiotic stresses. Micro Tom plants were imposed to anoxia and salinity stresses and photosynthetic parameters were used to evaluate the T6776 effects on plants recovery. Results show that T6776 is able to ameliorate plants stress tolerance affecting the photosynthetic response. Moreover, T6776 enhances plants emergency and resistance against important soil and air borne pathogens. Biological assays show a decrease in mortality of Micro Tom plants due to Fusarium oxysprum f. sp. radicis lycopersici, suggesting a protective role of seedlings by T6776. Similarly, the necrotic area caused on tomato leaves after Alternaria solani inoculation was reduced in T6776 treated plants, suggesting an induction of resistance by T6776. These preliminary results were discussed and

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the mechanisms involved were suggested, based also on previously collected information on the system T6776- Micro Tom. Plant root colonization seems to be a desired capability for a potential biocontrol strains, because some evidences correlate some positive plant response to root colonization ability. In order to investigate mechanisms involved in this process, in Chapter 5, we evaluate the involvement of LysM proteins in root colonization using the T6776-MicroTom system. LysM encoding genes in the T6776 genome where identified and the encoded LysM proteins characterized. The expression levels of the LysM secreted proteins encoding genes were measured by qPCR after 24 hours post T6776 inoculation on tomato roots. We show that T6776 encodes for eleven LysM proteins and, among them, six were predicted to be secreted. Among the secreted ones, the expression levels of four LysM encoding genes were found to increase in T6776 interacting with roots. The four induced genes encoded for similar genes products. Two genes (Th01572 and Th05014) encode for a subgroup C of chitinases, presenting the glycosyl hydrolases GH18 domain in addition to the two LysM domain, the other two genes (Th01574 and Th05012) encode for small LysM proteins, presenting only several number of LysM domains (3 and 4 respectively). Our results are the first insight into the possible involvement of these class of proteins in a beneficial organism - plant interaction, even if more studies are needed for elucidate their involvement in plant root colonization by T6776. Finally, in Chapter 6 a general discussion of the main results obtained in this PhD research work was provided. The discussion is accompanied by personal suggestion on a possible future research using the T6776-MicroTom system set up during this work.

Bibliography

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Baroncelli R, Piaggeschi G, Fiorini L, et al (2015) Draft Whole-Genome Sequence of the Biocontrol Agent Trichoderma harzianum T6776. 3:9–10. doi: 10.1128/genomeA.00647-15.Copyright

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Brondi D., BSc Thesis (2008/2009). Endofitismo di Trichoderma spp. in Solanum lycpersicum. Università di Pisa.

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Compant, S., Clément, C., & Sessitsch, A. (2010). Plant growth-promoting bacteria in the rhizo-and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biology and Biochemistry, 42(5), 669-678.

Campos ML, Carvalho RF, Benedito VA, Peres LEP (2010) Small and remarkable: The Micro-Tom model system as a tool to discover novel hormonal functions and interactions. Plant Signal Behav 5:267–270. doi: 10.4161/psb.5.3.10622

Carvalho RF, Campos ML, Pino LE, et al (2011) Convergence of developmental mutants into a single tomato model system: “Micro-Tom” as an effective toolkit for plant development research. Plant Methods 7:18. doi: 10.1186/1746-4811-7-18

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Fiorini L, Guglielminetti L, Mariotti L, et al (2015) Trichoderma harzianum T6776 modulates a complex metabolic network to stimulate tomato cv. Micro-Tom growth. Plant Soil 400:1–16. doi: 10.1007/s11104-015-2736-6

Flint, M. L., & Van Den Bosch, R. (1981). Introduction to Integrated Pest ManagementPlenum Press. New York. Glare T, Caradus J, Gelernter W, et al (2012) Have biopesticides come of age? Trends Biotechnol 30:250–258.

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CHAPTER 2: T6776 species assignment by phylogenetic analysis

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Chapter 2 WHEN A BIOCONTROL ISOLATE BELONGS TO A

SPECIES COMPLEX : T6776 SPECIES ASSIGNEMENT

BY PHYLOGENETIC ANALYSIS.

Abstract

Species identification is based on different methods that provide different level of accuracy: the advent of the genomic era provides additional tools for species identification, leading to a re-classification of several genera and species. T. harzianum has been defined as a species complex, including more than 15 species and several new species have been recognized. The isolate T6776 of Trichoderma used in this work has been previously identified as T. harzianum based on morphology and ITS sequencing. In order to define the placement of the isolate T6776 within the T. harzianum species complex a phylogenetic analysis was performed by using the sequence of five unlinked genes available in public database (act, cal, its, rpb2, tef1). Twenty-nine isolates belonging to the T. harzianum species complex were selected for the analysis and T. virens was used as out-group. The availability of the T6776 genome sequence facilitated the phylogenetic analysis, since genes sequences were already available. Results showed that T6776 belongs to T. afroharzianum species, the tropical T. harzianum sensu lato mainly isolated in African regions. Interestingly, two commercial biocontrol isolate, "T. harzianum T22" and the active ingredient of Canna AkTRIvator® (G.J.S. 08-137) were recently revised as T. afroharzianum isolates by Chaverri et al. (2015).

1. Introduction

1.1 Species concept within the Trichoderma genus

Members of this genus were mostly identified on the basis of morphology according to the MSR (Morphological Species Recognition) concept, since the BSR (Biological Species

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Recognition) seems not to be applicable to Trichoderma, as all but T. reesei isolates belonging to this genus apparently cannot be crossed so far. However, the use of ITS1 and ITS2 provides only poor phylogenetic resolution in some clades, as Pachybasium B clade (Kullnig-Gradinger et al., 2002; Chaverri et al., 2003a). Thus, researchers use several unlinked genes for phylogenetic studies following the GCPSR concept (Genealogical Concordance Phylogenetic Species Recognition) (Taylor et al., 2000). The simultaneous usage of several unlinked genes as diagnostic regions may lead to the most reliable phylogeny.

1. 2 The Trichoderma harzianum species complex

The holomorphic fungus T. harzianum is usually asexual (or anamorphic) and belong to the genus Trichoderma of the order Hypocreales (Ascomycota), which comprises a large number of species with a worldwide distribution (Bissett et al., 2003). T. harzianum, has been known for a long time as a single species (T. harzianum sensu lato) and with this meaning it can be considered as the most common and widespread Trichoderma species, due to the fact that it is the most frequent Trichoderma sp. recovered from samples world-wide (Druzhinina et al., 2005; Migheli et al., 2009). Thus, it has often been synonymized with Trichoderma biocontrol agent in general (Hanson JR, 2005), since it is the active ingredient in several commercially available biofungicide and biofertilizer formulations (Kaewchai et al., 2009; Samuels and Hebbar, 2015). Isolates belonging to this species display a remarkable diversity of lifestyles ranging from saprotrophy in free soil and dead wood, in rhizosphere and on dead fungal biomass. Some isolates were found to be opportunistic human pathogen (Sandoval-Denis et al., 2014). Interestingly, a rhizosphere-competent T. harzianum may not only grow on plant root, but it could also be able to penetrate into root epidermis, interacting intimately with the plant (Alonso-Ramírez et al. 2014). Moreover, it is one of the species most commonly isolated as endophytes in the sapwood of tropical trees (Evans et al., 2003; Gazis and Chaverri, 2010; Drunzhinina et al., 2011; Chaverri and Samuels, 2013). T. harzianum was one of the nine "aggregate species" recognized by Rifai (1969), which suggested that each aggregate species can include two or more distinct species. The T. harzianum sensu lato comprises the genetically distinct temperate agamospecies belonging to the group of closely related, albeit diverse, species of the Harzianum clade of Trichoderma (Hypocreales,

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Ascomycota; Dikarya). This species complex has been proposed to be a complex of several cryptic species (Druzhinina et al. 2010) and, recently, the complexity of this species has been studied by Chaverri et al. (2015) using a multilocus phylogenetic approach. This study revised the species complex defining the names of the species, which are at least 15. Nine species are described as new: T. afarasin, T. afroharzianum, T. abtrobrunneum, T. camerunense, T. endophyticum, T. neotropicale, T. pyramidale, T. rifaii and T. simmonsii. Since our Trichoderma isolate T6776 was firstly identified as a T. harzianum both morphologically and by ITS sequence (NCBI Accession Number GU997086), we made a phylogenetic analysis using a multilocus approach for the identification and characterization of this isolate.

2. Materials and methods

2.1 Bioinformatics and phylogenetic analysis

In order to define the placement of the isolate T6776 within the T. harzianum species complex we performed a phylogenetic analysis using act, cal, its, rpb2 and tef1 sequences. Twenty-nine isolates (see Table 2.1), one representative for each species, belonging to the Trichoderma harzianum species complex were selected for the analysis based of the epytype and on the number of available genes in public database according to Chaverri et al. (2015). Trichoderma virens isolates were used as out-group.

Multiple sequence alignments were exported to MEGA5 (Tamura et al. 2011) where best-fit substitution models were calculated for each separate sequence dataset. When necessary, in order to evaluate whether the five loci sequenced were congruent and suitable for concatenation, tree topologies of 75% Neighbour-Joining bootstrap and maximum parsimony analysis (100,000 replicates) were separately performed on gene and visually compared (Mason-Gamer et al. 1996). The multilocus concatenation alignment (act, cal, its, rpb2 and tef1) was performed with Geneious 8.1 created by Biomatters and available from http://www.geneious.com/.

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A Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities using MrBayes 3.2.1 (Ronquist and Huelsenbeck 2003) for single set of data as well as combined sequence datasets. Models of nucleotide substitution for each gene determined by MEGA5 were included for each locus. The analyses of two MCMC chains were run from random trees for numbers of generations necessary to reach 0,01 and sampled every 100 generations.

Table 2.1: Strains of Harzianum complex used in the phylogenetic analysis for the identification of T6776 species. In the table were reported geographic origin, the substrate from which the strain was isolated and the accession number for each gene (act, cal, its, rpb2 and tef1) following Chaverri et al., 2015.

Species Voucher Geographicorigin Substrate act cal ITS rpb2 tef1 Reference T. aggressivum CBS 100525 UK Mushrum compost FJ442433AF442859AF057600 AF545541 AF348095 Chaverri et al. 2015 T. velutinum DAOM230013Nepal SOIL JN133614JN133550AF149873 JN133569 AY937415 Chaverri et al. 2015 T. simmonsii CBS 130431 USA, Maryland decaying bark FJ442526AF442869AF443917 FJ442757 AF443935 Chaverri et al. 2015 T. lixii CBS 110080 Thailand decayed Ganoderma basidiocarp FJ442533AF442872AF443920 AF443938 Chaverri et al. 2015 T. guizhouense IMI 374787 Indonesia Dead wood FJ442523AF442881AF443923 FJ442718 AF443941 Chaverri et al. 2015 T. atrobrunneum CBS 130429 Germany Decaying Pinus sylvestris FJ442525AF442886AF443926 FJ442735 AF443943 Chaverri et al. 2015 T. harzianum CBS 226.95 UK SOIL FJ442567AF442864AJ222720 AF545549 AF348101 Chaverri et al. 2015 T. camerunese CBS 137272 Cameroon SOIL FJ442537AF442875AY027780 AF348107 Chaverri et al. 2015 T. afroharzianum IMI 393972 Cameroon SOIL FJ442535AF442882AY027781 AF348106 Chaverri et al. 2015 T. tawa CBS 114233 Thailand dacaying bark FJ442570FJ442406AY737756 AY391956 FJ463313 Chaverri et al. 2015 T. cinnamomeum G.J.S. 97-237 USA, Missouri decaying wood FJ442582JN133524AY737759 AY391920 AY737732 Chaverri et al. 2015 T. stramineum CBS 114248 Sri Lanka decaying wood FJ442583FJ442386AY737765 AY391945 AY737746 Chaverri et al. 2015 T. catoptron CBS 114232 Sri Lanka decaying wood FJ442584FJ442387AY737766 AY391900 AY737726 Chaverri et al. 2015 T. tomentosum DAOM178713aCanada, Ontario Ulmus wood JN133612JN133548EU330958AF545557 AY750882 Chaverri et al. 2015 T. alni CBS 120633 UK, England Alnus glutinosa wood EU498326EU518651EU498349EU498312Chaverri et al. 2015 T. brunneoviride CBS 120928 AUSTRIA Alnus incana wood EU498330EU518661EU498358EU498318Chaverri et al. 2015 T. endophyticum CBS 130730 Ecuador Theobroma gileri trunk endophyteFJ442446FJ442293FJ442242 FJ442721 FJ463314 Chaverri et al. 2015 T. lentiforme CBS 130744 Ecuador Theobroma cacao trunk endophyteFJ442470FJ442314FJ442271 FJ442791 FJ463359 Chaverri et al. 2015 T. rifaii CBS 130745 Panama Theobroma cacao trunk endophyteFJ442471FJ442315FJ442621 FJ442720 FJ463321 Chaverri et al. 2015 T. afarasin Dis377a Cameroon Cola sp. trunk endophytes FJ442562KP115277FJ442665 FJ442799 FJ463322 Chaverri et al. 2015 T. dacrymycellum WU 29044 Germany decaying wood FJ860749 FJ860533 FJ860633 Chaverri et al. 2015 T. parepimyces CBS 122769 AUSTRIA decorticated brunch of Pagus sylvatica FJ860800 FJ860562 FJ860664 Chaverri et al. 2015 T. amazonicum CBS 126898 Peru Hevea brasiliensis trunk endophyteKC561140 HM142358HM142367HM142376Chaverri et al. 2015 T. pleuroticola CBS 124383 Korea Pleurotus substrate JN133598JN133538HM142362HM142371HM142381Chaverri et al. 2015 T. pleuroti CBS 124387 Korea Pleurotus substrate JN133599JN133539HM142363HM142372HM142382Chaverri et al. 2015 T. neotropicale G.J.S. 11-185 Peru Hevea guianensis trunk endophyteKP115268KP115279HQ022407 HQ022771Chaverri et al. 2015 T. ceraceum G.J.S. 88-28 USA, New York decaying wood AY391901 AY391964 Chaverri et al. 2015 T. compactum CBS 121218 China SOIL KP115276 AY941824 Chaverri et al. 2015 T. pyramidale CBS 135574 Italy Olea europaea KJ665334 KJ665699 Chaverri et al. 2015

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3. Results

3.1 Bioinformatics and phylogenetic analysis: T6776 species identification

Five unlinked genes sequence from 31 Trichoderma isolates (29 ephytype isolates, T6776 isolate and T. virens as outgroup) were used to construct a phylogenetic tree, in order to recognized T6776 at species level. The results show that T6776 cluster with T. afroharzianum (Figure 1), the tropical strains mainly isolated in African regions. Interestingly, two commercial biocontrol isolate, "T. harzianum T22" and the active ingredient of Canna AkTRIvator® (G.J.S. 08-137) were recently revised as T. afroharzianum by Chaverri et al. (2015). Authors reported that T. afroharzianum was isolated mostly from soil, as our T6776 isolate.

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Figure 2.1: Bayesian MCMC analysis tree constructed from the alignment based on the concatenation of act, cal, its, rpb2 and tef1 partial sequences of 29 Trichoderma harzianum species complex isolates. The tree was rooted with sequences from T. virens. Only branches with a posterior probability of at least 0.95.

4. Discussion

In this chapter we identify the correct species of the isolate used in the present work, interesting for its growth promotion and biocontrol abilities. The availability of the T6776 genome sequence facilitated the phylogenetic analysis, since genes sequences were already available. As shown in this chapter, "T. harzianum T6776" has to be assigned to the T. afroharzianum species, as other patented biocontrol isolate re-identified by Chaverri et al. (2015). Accordingly to what reported by Chaverri et al., 2015, isolates belonging to this

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species were frequently isolated from soil sample, as our T6776 that was isolated from pinewood soil near Pisa, Italy. The correct species identification of a potentially beneficial isolate is very important for its application. The identification of a species has a major impact for manufacturers and users of the T. harzianum-based biopesticides, to avoid the risks of the application of potentially unsafe biocontrol agents. In fact, some members of the T. brevicompactum clade are able to produce trichodermin, which is a potent mycotoxin, and were often misidentified as T. harzianum (Chaverri et al. 2015). Moreover, it is known that within Trichoderma genus, morphologically identical, phylogenetic sister species express different biological properties (Druzhinina et al. 2010). The assignment of our isolate to the T. afroharzianum species suggests that isolates belonging to it, share common physiological features making them beneficial to plants as growth promoter or as biocontrol agent.

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