Exploring the endophytic fungal microbiome of tomato roots: The role of phytohormones in plant-endophyte interactions

Andrea Manzotti

Exploring the endophytic fungal microbiome of tomato roots: The role of phytohormones in plant-endophyte interactions

Andrea Manzotti

Abstract

The interactions between plants and microorganisms have been studied for decades and in recent years the beneficial effects conferred by certain microbial species to the host plant have been exploited as a sustainable solution for yield improvement in agriculture. Among the plethora of microorganisms constantly interacting with the plant, endophytes represent the fraction living asymptomatically inside the host tissues. In this PhD project, I investigated the interaction between tomato plants and fungal endophytes, studying both the root endophytic microbiome and the interaction between plants and single fungal endophyte species, mainly focusing on the role of hostgenotype and phytohormones in the interaction. In Manuscript I, we studied the role of host genotype and phytohormones on the structure of the fungal endophytic communities in tomato roots by amplicon sequencing and combined this approach with isolation and functional characterization of fungal endophytes. The amplicon sequencing analysis of the ITS1 region on the endophytic communities of two tomato genotypes (Castlemart and UC82B) revealed that the host genotype strongly affects the composition of the microbiome. Surprisingly, root pathogens were highly abundant in both genotypes and the most prevalent pathogenic taxa differed significantly in their relative abundance between the two genotypes. In contrast, the amplicon sequencing analysis on the fungal endophytic communities of two tomato mutant lines impaired in jasmonic acid synthesis (def1) and ethylene accumulation (line 8338) showed only minor changes in the composition of the microbiome compared to the respective wild types (Castlemart and UC82B, respectively). However, the species richness in def1 roots was significantly higher compared to its wild type. Moreover, the analysis of the phytohormone profilesof the two mutant lines and their respective wild types showed that the lower levels of jasmonic acid and ethylene, respectively, resulted in changes of the total phytohormone balance in both leaves and roots. This indicates that the higher species richness in the def1 mutant may be the result of more complex changes in the hormone signalling network. In the same study we also performed isolation and functional characterization of the endophytes showing that healthy tomato roots harbour a complex fungal community made of beneficial, commensal and pathogenic species. The 107 endophytic fungi isolated were classified in 18 different species, and the isolates identified as Colletotrichum coccodes, Plectosphaerella sp., Pyrenochaeta lycopersici, Thielaviopsis basicola, Alternaria infectoria and Fusarium sp. showed a pathogenic lifestyle when applied individually to tomato seedlings, although they were originally isolated from roots of healthy plants. In contrast, the remaining 12 species showed either asymptomatic or beneficial behaviour. Collectively, the combined results of the cultivationdependentand cultivation-independent approaches strongly suggest that the interactions between members of the endophytic microbiome may sustain the equilibrium in the community and prevent high abundant pathogenic taxa from causing diseases. In Manuscript II, the role of phytohormones on plant-endophyte interactions was investigated in the interaction between tomato roots and the Mucoromycotina fungus Umbelopsis isabellina, isolated during the characterization of the root microbiome. Initially, qPCR was used to examine the ability of U. isabellina to colonize roots of different tomato mutant lines impaired in biosynthesis of abscisic acid (notabilis) and jasmonic acid (def1) or in accumulation of ethylene (line 8338) and salicylic acid (NahG). Lower levels of abscisic acid and ethylene resulted in reduced fungal colonization of the roots compared to the wild types, whereas lower levels of jasmonic acid and salicylic acid resulted in increased root colonization by the fungus. In order to go deeper into the role of abscisic acid in this interaction, we performed an RNA sequencing analysis on the tomato mutant notabilis and its wild type Ailsa Craig with and without U. isabellina inoculation. Defence-related genes, such as phytohormone and specialized metabolite biosynthesis genes were down-regulated in the roots of the wild type during the interaction with the endophyte, highlighting the need of a general suppression of the host immune responses during the interaction. In contrast, the comparison of the root transcriptome of notabilis and its wild type during U. isabellina colonization showed a significantly higher expression of ethylene and salicylic acid signalling genes in the mutant line. This strongly suggests that the reduction in fungal colonization of notabilis roots compared to the wild type may be linked to the stronger defence responses in the mutant line. Furthermore, we showed that the beneficial effects conferred by U. isabellina to the host plant are reduced in the abscisic acid mutant notabilis compared to its wildtype. Collectively, these results highlight the importance of this hormone in the establishment of the beneficial interaction between tomato roots and U. isabellina.

Originalsprog	Engelsk

Forlag	Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen
Status	Udgivet - 2019

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@phdthesis{9752ade80bf5428cbda4fbda44d79e74,

title = "Exploring the endophytic fungal microbiome of tomato roots: The role of phytohormones in plant-endophyte interactions",

abstract = "The interactions between plants and microorganisms have been studied for decades and in recent years the beneficial effects conferred by certain microbial species to the host plant have been exploited as a sustainable solution for yield improvement in agriculture. Among the plethora of microorganisms constantly interacting with the plant, endophytes represent the fraction living asymptomatically inside the host tissues. In this PhD project, I investigated the interaction between tomato plants and fungal endophytes, studying both the root endophytic microbiome and the interaction between plants and single fungal endophyte species, mainly focusing on the role of hostgenotype and phytohormones in the interaction. In Manuscript I, we studied the role of host genotype and phytohormones on the structure of the fungal endophytic communities in tomato roots by amplicon sequencing and combined this approach with isolation and functional characterization of fungal endophytes. The amplicon sequencing analysis of the ITS1 region on the endophytic communities of two tomato genotypes (Castlemart and UC82B) revealed that the host genotype strongly affects the composition of the microbiome. Surprisingly, root pathogens were highly abundant in both genotypes and the most prevalent pathogenic taxa differed significantly in their relative abundance between the two genotypes. In contrast, the amplicon sequencing analysis on the fungal endophytic communities of two tomato mutant lines impaired in jasmonic acid synthesis (def1) and ethylene accumulation (line 8338) showed only minor changes in the composition of the microbiome compared to the respective wild types (Castlemart and UC82B, respectively). However, the species richness in def1 roots was significantly higher compared to its wild type. Moreover, the analysis of the phytohormone profilesof the two mutant lines and their respective wild types showed that the lower levels of jasmonic acid and ethylene, respectively, resulted in changes of the total phytohormone balance in both leaves and roots. This indicates that the higher species richness in the def1 mutant may be the result of more complex changes in the hormone signalling network. In the same study we also performed isolation and functional characterization of the endophytes showing that healthy tomato roots harbour a complex fungal community made of beneficial, commensal and pathogenic species. The 107 endophytic fungi isolated were classified in 18 different species, and the isolates identified as Colletotrichum coccodes, Plectosphaerella sp., Pyrenochaeta lycopersici, Thielaviopsis basicola, Alternaria infectoria and Fusarium sp. showed a pathogenic lifestyle when applied individually to tomato seedlings, although they were originally isolated from roots of healthy plants. In contrast, the remaining 12 species showed either asymptomatic or beneficial behaviour. Collectively, the combined results of the cultivationdependentand cultivation-independent approaches strongly suggest that the interactions between members of the endophytic microbiome may sustain the equilibrium in the community and prevent high abundant pathogenic taxa from causing diseases. In Manuscript II, the role of phytohormones on plant-endophyte interactions was investigated in the interaction between tomato roots and the Mucoromycotina fungus Umbelopsis isabellina, isolated during the characterization of the root microbiome. Initially, qPCR was used to examine the ability of U. isabellina to colonize roots of different tomato mutant lines impaired in biosynthesis of abscisic acid (notabilis) and jasmonic acid (def1) or in accumulation of ethylene (line 8338) and salicylic acid (NahG). Lower levels of abscisic acid and ethylene resulted in reduced fungal colonization of the roots compared to the wild types, whereas lower levels of jasmonic acid and salicylic acid resulted in increased root colonization by the fungus. In order to go deeper into the role of abscisic acid in this interaction, we performed an RNA sequencing analysis on the tomato mutant notabilis and its wild type Ailsa Craig with and without U. isabellina inoculation. Defence-related genes, such as phytohormone and specialized metabolite biosynthesis genes were down-regulated in the roots of the wild type during the interaction with the endophyte, highlighting the need of a general suppression of the host immune responses during the interaction. In contrast, the comparison of the root transcriptome of notabilis and its wild type during U. isabellina colonization showed a significantly higher expression of ethylene and salicylic acid signalling genes in the mutant line. This strongly suggests that the reduction in fungal colonization of notabilis roots compared to the wild type may be linked to the stronger defence responses in the mutant line. Furthermore, we showed that the beneficial effects conferred by U. isabellina to the host plant are reduced in the abscisic acid mutant notabilis compared to its wildtype. Collectively, these results highlight the importance of this hormone in the establishment of the beneficial interaction between tomato roots and U. isabellina. ",

author = "Andrea Manzotti",

year = "2019",

language = "English",

publisher = "Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen",

}

TY - BOOK

T1 - Exploring the endophytic fungal microbiome of tomato roots

T2 - The role of phytohormones in plant-endophyte interactions

AU - Manzotti, Andrea

PY - 2019

Y1 - 2019

N2 - The interactions between plants and microorganisms have been studied for decades and in recent years the beneficial effects conferred by certain microbial species to the host plant have been exploited as a sustainable solution for yield improvement in agriculture. Among the plethora of microorganisms constantly interacting with the plant, endophytes represent the fraction living asymptomatically inside the host tissues. In this PhD project, I investigated the interaction between tomato plants and fungal endophytes, studying both the root endophytic microbiome and the interaction between plants and single fungal endophyte species, mainly focusing on the role of hostgenotype and phytohormones in the interaction. In Manuscript I, we studied the role of host genotype and phytohormones on the structure of the fungal endophytic communities in tomato roots by amplicon sequencing and combined this approach with isolation and functional characterization of fungal endophytes. The amplicon sequencing analysis of the ITS1 region on the endophytic communities of two tomato genotypes (Castlemart and UC82B) revealed that the host genotype strongly affects the composition of the microbiome. Surprisingly, root pathogens were highly abundant in both genotypes and the most prevalent pathogenic taxa differed significantly in their relative abundance between the two genotypes. In contrast, the amplicon sequencing analysis on the fungal endophytic communities of two tomato mutant lines impaired in jasmonic acid synthesis (def1) and ethylene accumulation (line 8338) showed only minor changes in the composition of the microbiome compared to the respective wild types (Castlemart and UC82B, respectively). However, the species richness in def1 roots was significantly higher compared to its wild type. Moreover, the analysis of the phytohormone profilesof the two mutant lines and their respective wild types showed that the lower levels of jasmonic acid and ethylene, respectively, resulted in changes of the total phytohormone balance in both leaves and roots. This indicates that the higher species richness in the def1 mutant may be the result of more complex changes in the hormone signalling network. In the same study we also performed isolation and functional characterization of the endophytes showing that healthy tomato roots harbour a complex fungal community made of beneficial, commensal and pathogenic species. The 107 endophytic fungi isolated were classified in 18 different species, and the isolates identified as Colletotrichum coccodes, Plectosphaerella sp., Pyrenochaeta lycopersici, Thielaviopsis basicola, Alternaria infectoria and Fusarium sp. showed a pathogenic lifestyle when applied individually to tomato seedlings, although they were originally isolated from roots of healthy plants. In contrast, the remaining 12 species showed either asymptomatic or beneficial behaviour. Collectively, the combined results of the cultivationdependentand cultivation-independent approaches strongly suggest that the interactions between members of the endophytic microbiome may sustain the equilibrium in the community and prevent high abundant pathogenic taxa from causing diseases. In Manuscript II, the role of phytohormones on plant-endophyte interactions was investigated in the interaction between tomato roots and the Mucoromycotina fungus Umbelopsis isabellina, isolated during the characterization of the root microbiome. Initially, qPCR was used to examine the ability of U. isabellina to colonize roots of different tomato mutant lines impaired in biosynthesis of abscisic acid (notabilis) and jasmonic acid (def1) or in accumulation of ethylene (line 8338) and salicylic acid (NahG). Lower levels of abscisic acid and ethylene resulted in reduced fungal colonization of the roots compared to the wild types, whereas lower levels of jasmonic acid and salicylic acid resulted in increased root colonization by the fungus. In order to go deeper into the role of abscisic acid in this interaction, we performed an RNA sequencing analysis on the tomato mutant notabilis and its wild type Ailsa Craig with and without U. isabellina inoculation. Defence-related genes, such as phytohormone and specialized metabolite biosynthesis genes were down-regulated in the roots of the wild type during the interaction with the endophyte, highlighting the need of a general suppression of the host immune responses during the interaction. In contrast, the comparison of the root transcriptome of notabilis and its wild type during U. isabellina colonization showed a significantly higher expression of ethylene and salicylic acid signalling genes in the mutant line. This strongly suggests that the reduction in fungal colonization of notabilis roots compared to the wild type may be linked to the stronger defence responses in the mutant line. Furthermore, we showed that the beneficial effects conferred by U. isabellina to the host plant are reduced in the abscisic acid mutant notabilis compared to its wildtype. Collectively, these results highlight the importance of this hormone in the establishment of the beneficial interaction between tomato roots and U. isabellina.

AB - The interactions between plants and microorganisms have been studied for decades and in recent years the beneficial effects conferred by certain microbial species to the host plant have been exploited as a sustainable solution for yield improvement in agriculture. Among the plethora of microorganisms constantly interacting with the plant, endophytes represent the fraction living asymptomatically inside the host tissues. In this PhD project, I investigated the interaction between tomato plants and fungal endophytes, studying both the root endophytic microbiome and the interaction between plants and single fungal endophyte species, mainly focusing on the role of hostgenotype and phytohormones in the interaction. In Manuscript I, we studied the role of host genotype and phytohormones on the structure of the fungal endophytic communities in tomato roots by amplicon sequencing and combined this approach with isolation and functional characterization of fungal endophytes. The amplicon sequencing analysis of the ITS1 region on the endophytic communities of two tomato genotypes (Castlemart and UC82B) revealed that the host genotype strongly affects the composition of the microbiome. Surprisingly, root pathogens were highly abundant in both genotypes and the most prevalent pathogenic taxa differed significantly in their relative abundance between the two genotypes. In contrast, the amplicon sequencing analysis on the fungal endophytic communities of two tomato mutant lines impaired in jasmonic acid synthesis (def1) and ethylene accumulation (line 8338) showed only minor changes in the composition of the microbiome compared to the respective wild types (Castlemart and UC82B, respectively). However, the species richness in def1 roots was significantly higher compared to its wild type. Moreover, the analysis of the phytohormone profilesof the two mutant lines and their respective wild types showed that the lower levels of jasmonic acid and ethylene, respectively, resulted in changes of the total phytohormone balance in both leaves and roots. This indicates that the higher species richness in the def1 mutant may be the result of more complex changes in the hormone signalling network. In the same study we also performed isolation and functional characterization of the endophytes showing that healthy tomato roots harbour a complex fungal community made of beneficial, commensal and pathogenic species. The 107 endophytic fungi isolated were classified in 18 different species, and the isolates identified as Colletotrichum coccodes, Plectosphaerella sp., Pyrenochaeta lycopersici, Thielaviopsis basicola, Alternaria infectoria and Fusarium sp. showed a pathogenic lifestyle when applied individually to tomato seedlings, although they were originally isolated from roots of healthy plants. In contrast, the remaining 12 species showed either asymptomatic or beneficial behaviour. Collectively, the combined results of the cultivationdependentand cultivation-independent approaches strongly suggest that the interactions between members of the endophytic microbiome may sustain the equilibrium in the community and prevent high abundant pathogenic taxa from causing diseases. In Manuscript II, the role of phytohormones on plant-endophyte interactions was investigated in the interaction between tomato roots and the Mucoromycotina fungus Umbelopsis isabellina, isolated during the characterization of the root microbiome. Initially, qPCR was used to examine the ability of U. isabellina to colonize roots of different tomato mutant lines impaired in biosynthesis of abscisic acid (notabilis) and jasmonic acid (def1) or in accumulation of ethylene (line 8338) and salicylic acid (NahG). Lower levels of abscisic acid and ethylene resulted in reduced fungal colonization of the roots compared to the wild types, whereas lower levels of jasmonic acid and salicylic acid resulted in increased root colonization by the fungus. In order to go deeper into the role of abscisic acid in this interaction, we performed an RNA sequencing analysis on the tomato mutant notabilis and its wild type Ailsa Craig with and without U. isabellina inoculation. Defence-related genes, such as phytohormone and specialized metabolite biosynthesis genes were down-regulated in the roots of the wild type during the interaction with the endophyte, highlighting the need of a general suppression of the host immune responses during the interaction. In contrast, the comparison of the root transcriptome of notabilis and its wild type during U. isabellina colonization showed a significantly higher expression of ethylene and salicylic acid signalling genes in the mutant line. This strongly suggests that the reduction in fungal colonization of notabilis roots compared to the wild type may be linked to the stronger defence responses in the mutant line. Furthermore, we showed that the beneficial effects conferred by U. isabellina to the host plant are reduced in the abscisic acid mutant notabilis compared to its wildtype. Collectively, these results highlight the importance of this hormone in the establishment of the beneficial interaction between tomato roots and U. isabellina.

UR - https://rex.kb.dk/permalink/f/h35n6k/KGL01012069452

M3 - Ph.D. thesis

BT - Exploring the endophytic fungal microbiome of tomato roots

PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen

ER -