Discovery and Transcriptional Profiling of Penicillium digitatum Genes That Could Promote Fungal Virulence during Citrus Fruit Infection.

Green mold caused by Penicillium digitatum (Pers.:Fr.) Sacc is the most prevalent postharvest rot concerning citrus fruits. Using the subtractive suppression hybridization (SSH) technique, different P. digitatum genes have been identified that could be involved in virulence during citrus infection in the early stages, a crucial moment that determines whether the infection progresses or not. To this end, a comparison of two P. digitatum strains with high and low virulence has been carried out. We conducted a study on the gene expression profile of the most relevant genes. The results indicate the importance of transcription and regulation processes as well as enzymes involved in the degradation of the plant cell wall. The most represented expressed sequence tag (EST) was identified as PDIP_11000, associated with the FluG domain, which is putatively involved in the activation of conidiation. It is also worth noting that PDIP_02280 encodes a pectin methyl esterase, a cell wall remodeling protein with a high expression level in the most virulent fungal strains, which is notably induced during citrus infection. Furthermore, within the group with the greatest representation and showing significant induction in the early stages of infection, regulatory proteins (PDIP_68700, PDIP_76160) and a chaperone (PDIP_38040) stand out. To a lesser extent, but not less relevant, it is worth distinguishing different regulatory proteins and transcription factors, such as PDIP_00580, PDIP_49640 and PDIP_78930.

Exploring the Biocontrol Capability of Non-Mycotoxigenic Strains of Penicillium expansum.

Penicillium expansum is one the major postharvest pathogens of pome fruit during postharvest handling and storage. This fungus also produces patulin, which is a highly toxic mycotoxin that can contaminate infected fruits and their derived products and whose levels are regulated in many countries. In this study, we investigated the biocontrol potential of non-mycotoxigenic strains of Penicillium expansum against a mycotoxigenic strain. We analyzed the competitive behavior of two knockout mutants that were unable to produce patulin. The first mutant (∆patK) involved the deletion of the patK gene, which is the initial gene in patulin biosynthesis. The second mutant (∆veA) involved the deletion of veA, which is a global regulator of primary and secondary metabolism. At the phenotypic level, the ∆patK mutant exhibited similar phenotypic characteristics to the wild-type strain. In contrast, the ∆veA mutant displayed altered growth characteristics compared with the wild type, including reduced conidiation and abnormal conidiophores. Neither mutant produced patulin under the tested conditions. Under various stress conditions, the ∆veA mutants exhibited reduced growth and conidiation when exposed to stressors, including cell membrane stress, oxidative stress, osmotic stress, and different pH values. However, no significant changes were observed in the ∆patK mutant. In competitive growth experiments, the presence of non-mycotoxigenic strains reduced the population of the wild-type strain during in vitro growth. Furthermore, the addition of either of the non-mycotoxigenic strains resulted in a significant decrease in patulin levels. Overall, our results suggest the potential use of non-mycotoxigenic mutants, particularly ∆patK mutants, as biocontrol agents to reduce patulin contamination in food and feed.

Ochratoxin A Defective Aspergillus carbonarius Mutants as Potential Biocontrol Agents.

Aspergillus carbonarius is one of the main species responsible for wine, coffee and cocoa toxin contamination. The main mycotoxin produced by this fungus, ochratoxin A (OTA), is a secondary metabolite categorized as a possible carcinogen because of its significant nephrotoxicity and immunosuppressive effects. A polyketide synthase gene (otaA) encodes the first enzyme in the OTA biosynthetic pathway. It is known that the filamentous fungi, growth, development and production of secondary metabolites are interconnected processes governed by global regulatory factors whose encoding genes are generally located outside the gene clusters involved in the biosynthesis of each secondary metabolite, such as the veA gene, which forms part of the VELVET complex. Different fungal strains compete for nutrients and space when they infect their hosts, and safer non-mycotoxigenic strains may be able to outcompete mycotoxigenic strains during colonization. To determine the possible utility of biopesticides based on the competitive exclusion of mycotoxigenic strains by non-toxigenic ones, we used A. carbonarius ΔotaA and ΔveA knockout mutants. Our results showed that during both in vitro growth and infection of grapes, non-mycotoxigenic strains could outcompete the wild-type strain. Additionally, the introduction of the non-mycotoxigenic strain led to a drastic decrease in OTA during both in vitro growth and infection of grapes.

Impact of the antifungal protein PgAFP on the proteome and patulin production of Penicillium expansum on apple-based medium.

Apples are prone to be contaminated with Penicillium expansum, which produces the mycotoxin patulin, posing a risk for human health. Antifungal treatments are required to control this fungal pathogen, although consumers demand products free of synthetic additives. Then, the use of antifungal proteins produced by moulds represents a novel and promising strategy. Although its inhibitory effect on P. expansum has been reported, the impact of these proteins on patulin production has been scarcely studied, pointing to a possible patulin overproduction. The aim of this work was to evaluate the effect of the antifungal protein PgAFP on the proteome and patulin biosynthesis of P. expansum grown in apple-based agar, intending to decipher these effects without the apple in vivo physiological response to the fungal infection. PgAFP increased the production of patulin on three of the five P. expansum strains evaluated. The proteome of the PgAFP-treated P. expansum showed five proteins involved in patulin biosynthesis in higher abundance (fold change 2.8-9.8), as well as proteins related to pathogenicity and virulence that suggest lower ability to infect fruits. Additionally, several proteins associated with oxidative stress, such as glutathione peroxidase, redoxin, or heat shock proteins were found in higher abundance, pointing to a response against oxidative stress elicited by PgAFP. These results provide evidence to be cautious in applying this antifungal protein in apples, being of utmost relevance to provide knowledge about the global response of P. expansum against an antifungal protein with many shared characteristics with others. These findings significantly contribute to future studies of assessment and suitability of not only these antifungal proteins but also new antifungal compounds.

Securing fruit production: Opportunities from the elucidation of the molecular mechanisms of postharvest fungal infections.

Fruit-based diets have been adopted by the public worldwide because of their nutritional value. Many advances have also been made in the elucidation of host-pathogen interaction in the postharvest phase of fruits, in the hope of improving the management of diseases caused by pathogenic molds. In this study, we presented the molecular mechanisms by which pathogenic mold infects fruit in the postharvest phase, and focused on the knowledge gained from recent molecular techniques such as differential analysis of gene expression, targeted insertion, and mutagenesis. Current postharvest pathogenic fungal control strategies were then examined on the basis of their mechanisms for altering the infection process in order to explore new perspectives for securing fruit production. We found that biotechnological advances have led to an understanding of the new basic molecular processes involved in fruit fungal infection and to the identification of new genes, proteins and key factors that could serve as ideal targets for innovative antifungal strategies. In addition, the most commonly used steps to evaluate an approach to disrupt the fruit fungal infection process are mainly based on the inhibition of mycelial growth, spore germination, disruption of Adenosine triphosphate (ATP) synthesis, induction of oxidative stress, cell wall membrane damage, and inhibition of key enzymes. Finally, the alteration of the molecular mechanisms of signaling and response pathways to infection stimulation should also guide the development of effective control strategies to ensure fruit production.

Functional Role of Aspergillus carbonariusAcOTAbZIP Gene, a bZIP Transcription Factor within the OTA Gene Cluster.

Aspergillus carbonarius is the principal fungal species responsible for ochratoxin A (OTA) contamination of grapes and derived products in the main viticultural regions worldwide. In recent years, co-expressed genes representing a putative-OTA gene cluster were identified, and the deletion of a few of them allowed the partial elucidation of the biosynthetic pathway in the fungus. In the putative OTA-gene cluster is additionally present a bZIP transcription factor (AcOTAbZIP), and with this work, A. carbonarius ΔAcOTAbZIP strains were generated to study its functional role. According to phylogenetic analysis, the gene is conserved in the OTA-producing fungi. A Saccharomyces cerevisiae transcription factor binding motif (TFBM) homolog, associated with bZIP transcription factors was present in the A. carbonarius OTA-gene cluster no-coding regions. AcOTAbZIP deletion results in the loss of OTA and the intermediates OTB and OTß. Additionally, in ΔAcOTAbZIP strains, a down-regulation of AcOTApks, AcOTAnrps, AcOTAp450, and AcOTAhal genes was observed compared to wild type (WT). These results provide evidence of the direct involvement of the AcOTAbZIP gene in the OTA biosynthetic pathway by regulating the involved genes. The loss of OTA biosynthesis ability does not affect fungal development as demonstrated by the comparison of ΔAcOTAbZIP strains and WT strains in terms of vegetative growth and asexual sporulation on three different media. Finally, no statistically significant differences in virulence were observed among ΔAcOTAbZIP strains and WT strains on artificially inoculated grape berries, demonstrating that OTA is not required by A. carbonarius for the pathogenicity process.

Coordinated activation of the metabolic pathways induced by LED blue light in citrus fruit.

The biochemical changes induced by LED Blue Light (LBL) (450 nm) in Lane Late oranges were investigated. The selected quantum flux (60 µmol m-2 s-1, 2 days) was associated with resistance against Penicillium digitatum, the main postharvest pathogen of citrus fruit. A holistic overview was obtained by a comparative transcriptome profile analysis, which revealed that LBL favored energy metabolism and redirected metabolic pathways toward the synthesis of diverse primary and secondary metabolism products. LBL favored reactive oxygen species homeostasis and metabolic activities involving lipid metabolism, specifically the synthesis of pigments and oxylipins, and the metabolism of carbohydrates, amino acids and indol- and alkaloid-derivatives. LBL also repressed limonene catabolism and triggered phenylpropanoid derivatives-related changes, which increased content in total flavonoids. Transferring fruit from LBL to darkness favored those processes involving amino acids, different phenylpropanoid, alkaloid and terpenoid classes, and ferrochelatase activity.

EFE-Mediated Ethylene Synthesis Is the Major Pathway in the Citrus Postharvest Pathogen Penicillium digitatum during Fruit Infection.

Penicillium digitatum is the main fungal postharvest pathogen of citrus fruit under Mediterranean climate conditions. The role of ethylene in the P. digitatum-citrus fruit interaction is unclear and controversial. We analyzed the involvement of the 2-oxoglutarate-dependent ethylene-forming enzyme (EFE)-encoding gene (efeA) of P. digitatum on the pathogenicity of the fungus. The expression of P. digitatumefeA parallels ethylene production during growth on PDA medium, with maximum levels reached during sporulation. We generated ΔefeA knockout mutants in P. digitatum strain Pd1. These mutants showed no significant defect on mycelial growth or sporulation compared to the parental strain. However, the knockout mutants did not produce ethylene in vitro. Citrus pathogenicity assays showed no differences in virulence between the parental and ΔefeA knockout mutant strains, despite a lack of ethylene production by the knockout mutant throughout the infection process. This result suggests that ethylene plays no role in P. digitatum pathogenicity. Our results clearly show that EFE-mediated ethylene synthesis is the major ethylene synthesis pathway in the citrus postharvest pathogen P. digitatum during both in vitro growth on PDA medium and the infection process, and that this hormone is not necessary for establishing P. digitatum infection in citrus fruit. However, our results also indicate that ethylene produced by P. digitatum during sporulation on the fruit surface may influence the development of secondary fungal infections.

Elaborated regulation of griseofulvin biosynthesis in Penicillium griseofulvum and its role on conidiation and virulence.

Penicilium griseofulvum, the causal agent of apple blue mold, is able to produce in vitro and on apple a broad spectrum of secondary metabolites (SM), including patulin, roquefortine C and griseofulvin. Among them, griseofulvin is known for its antifungal and antiproliferative activity, and has received interest in many sectors, from medicine to agriculture. The biosynthesis of SM is finely regulated by filamentous fungi and can involve global regulators and pathway specific regulators, which are usually encoded by genes present in the same gene cluster as the backbone gene and tailoring enzymes. In the griseofulvin gene cluster, two putative transcription factors were previously identified, encoded by genes gsfR1 and gsfR2, and their role has been investigated in the present work. Analysis of P. griseofulvum knockout mutants lacking either gene suggest that gsfR2 forms part of a different pathway and gsfR1 exhibits many spectra of action, acting as regulator of griseofulvin and patulin biosynthesis and influencing conidia production and virulence on apple. The analysis of gsfR1 promoter revealed that the regulation of griseofulvin biosynthesis is also controlled by global regulators in response to many environmental stimuli, such as carbon and nitrogen. The influence of carbon and nitrogen on griseofulvin production was further investigated and verified, revealing a complex network of response and confirming the central role of gsfR1 in many processes in P. griseofulvum.

Evaluation of the activity of the antifungal PgAFP protein and its producer mould against Penicillium spp postharvest pathogens of citrus and pome fruits.

Postharvest fungal diseases are among the main causes of fresh fruit losses. Chemical control is against claims for "natural" or "chemical-free" products. Biocontrol agents, such as antifungal proteins or their producing moulds, may serve to combat unwanted pathogens. Since the effectiveness of these bioprotective agents depends on the food substrate, their effect must be tested on fruits. The objective of this work was to study the effect of the antifungal protein PgAFP and its producer, Penicillium chrysogenum, against Penicillium expansum and Penicillium digitatum growth on apple and oranges respectively, and the PgAFP effect on eleven P. expansum, Penicillium italicum, and P. digitatum strains in vitro, and on patulin production on apple substrate. The sensitivity upon PgAFP was P. digitatum > P. expansum > P. italicum. In oranges, broadly, no inhibitory effect was obtained. PgAFP and P. chrysogenum did not inhibit the P. expansum CMP-1 growth on Golden Delicious apples, however, a successful effect was achieved on Royal Gala apples. On apple substrate, patulin production by P. expansum CMP-1 rose in parallel to PgAFP concentrations, linked with high reactive oxygen species levels. PgAFP cannot be proposed as a bioprotective agent on apple. However, P. chrysogenum is a promising agent to be used on Royal Gala apples.

Functional and Pharmacological Analyses of the Role of Penicillium digitatum Proteases on Virulence.

Penicillium digitatum is the major postharvest pathogen of citrus fruit under Mediterranean climate conditions. Previous results have shown that proteases is the largest enzyme family induced by P. digitatum during fruit infection. In the present work, we addressed the study of the role of P. digitatum's proteases in virulence following two complementary approaches. In the first approach, we undertook the functional characterization of the P. digitatum prtT gene, which codes for a putative transcription factor previously shown to regulate extracellular proteases in other filamentous fungi. Deletion of prtT caused a significant loss in secreted protease activity during in vitro growth assays. However, there was no effect on virulence. Gene expression of the two major secreted acid proteases was barely affected in the ΔprtT deletant during infection of citrus fruit. Hence, no conclusion could be drawn on the role of these secreted acidic proteases on the virulence of P. digitatum. In the second approach, we studied the effect of different protease inhibitors and chelators on virulence. Co-inoculation of citrus fruit with P. digitatum conidia and a cocktail of protease inhibitors resulted in almost a complete absence of disease development. Analysis of individual inhibitors revealed that the metalloprotease inhibitor, 1,10-phenanthroline, was responsible for the observed effect. The application of metal ions reverted the protective effect caused by the metallopeptidase inhibitor. These results may set the basis for the development of new alternative treatments to combat this important postharvest pathogen.

In-Depth Characterization of Bioactive Extracts from Posidonia oceanica Waste Biomass.

Posidonia oceanica waste biomass has been valorised to produce extracts by means of different methodologies and their bioactive properties have been evaluated. Water-based extracts were produced using ultrasound-assisted and hot water methods and classified according to their ethanol-affinity (E1: ethanol soluble; E2: non-soluble). Moreover, a conventional protocol with organic solvents was applied, yielding E3 extracts. Compositional and structural characterization confirmed that while E1 and E3 extracts were mainly composed of minerals and lipids, respectively, E2 extracts were a mixture of minerals, proteins and carbohydrates. All the extracts showed remarkably high antioxidant capacity, which was not only related to phenolic compounds but also to the presence of proteins and polysaccharides. All E2 and E3 extracts inhibited the growth of several foodborne fungi, while only E3 extracts decreased substantially the infectivity of feline calicivirus and murine norovirus. These results show the potential of P. oceanica waste biomass for the production of bioactive extracts.

Identification and Functional Analysis of NLP-Encoding Genes from the Postharvest Pathogen Penicillium expansum.

Penicillium expansum is a major postharvest pathogen that infects different fruits, mainly through injuries inflicted during harvest or subsequent handling after harvest. Several effectors were suggested to mediate pathogenicity of P. expansum in fruit tissue. Among these effectors Nep1-like proteins (NLPs), produced by various microorganisms with different lifestyles, are known for their ability to induce necrosis in dicot plants and were shown to be involved in virulence of several plant-related pathogens. This study was aimed at the identification and functional characterization of two NLP genes found in the genome of P. expansum. The genes were designated Penlp1 and Penlp2 and were found to code type1 and type3 NLP respectively. Necrosis-inducing activity of the two proteins was demonstrated by transient expression in Nicotiana benthamiana leaves. While Penlp1 expression was induced during apple infection and in liquid culture, the highest level of Penlp2 expression was found in ungerminated spores. Deletion of Penlp1, but not Penlp2, resulted in reduced virulence on apples manifested by reduced rate of lesion development (disease severity).

Differential contribution of the two major polygalacturonases from Penicillium digitatum to virulence towards citrus fruit.

The fungus Penicillium digitatum is the causal agent of the citrus green mould, the major postharvest diseases of citrus fruit. Lesions on the surface of infected fruits first appear as soft areas around the inoculation site, due to maceration of fruit. The macerating activity has been associated with pectinases secreted by the fungus during infection. In order to evaluate the contribution to virulence and macerating activity of the two major polygalacturonases (PGs) secreted by P. digitatum, we have obtained and characterized mutants lacking either pg1 or pg2, the genes encoding PG1 and PG2, respectively. Disease incidence of deletants in either gene was not different from that of the parental strain or ectopic transformants. However, disease progressed more slowly in deletants, especially in those lacking the pg2 gene. The lesions originated by the Δpg2 deletants were not as soft and the pH was not as acid as those originated by either the wild type strain or the ectopic transformants. Total PG activity in the macerated tissue was also lower in fruits infected with the Δpg2 deletants. Interestingly, the macerated tissue of oranges infected with Δpg2 deletants showed around 50% reduction in galacturonic acid content with respect to lesions caused by any other strain.

Functional Characterization of the alb1 Orthologue Gene in the Ochratoxigenic Fungus Aspergillus carbonarius (AC49 strain).

Aspergillus carbonarius, belonging to the group Nigri, is the main species responsible for contamination by ochratoxin A (OTA) in grapes and derivative products. OTA can accumulate in the mycelium and in black conidia of the fungus and released into the matrix. Here, we have deleted in A. carbonarius the alb1 orthologue gene of A. fumigatus, involved in melanin biosynthesis. Three A. carbonarius Δalb1 mutants were characterized for morphologic traits and OTA production on different media and temperatures. Δalb1 mutants showed a fawn color of conidia associated with a significant reduction of the conidiogenesis and a statistically significant increase (p ≤ 0.01) of total OTA production as compared to the wild type (WT) strain. The alb1 gene somehow affected OTA partitioning since in Δalb1 mutants OTA amount was lower in conidia and was more abundantly secreted into the medium as compared to the WT. On grape berries the Δalb1 mutants and the WT caused lesions with similar sizes but OTA amount in berry tissues was higher for the mutants. These results demonstrate that A. carbonarius conidia pigmentation is largely dependent on polyketide biosynthesis. The gene is not directly involved in virulence and its deletion affects morphological features and OTA production in the fungus.

Unravelling the contribution of the Penicillium expansum PeSte12 transcription factor to virulence during apple fruit infection.

Blue mould disease caused by Penicillium expansum infection is one of the most important diseases of pome fruit accounting for important economic losses. In the present study, the PeSte12 transcription factor gene was identified, and deletant mutants were produced by gene replacement. Knockout mutants showed a significant decrease of virulence during apple fruit infection. Virulence was affected by the maturity stage of the fruit (immature, mature and over-mature), and disease severity was notably reduced when the apples were stored at 0 °C. The ΔPeSte12 mutants resulted defective in asexual reproduction, producing less conidia, but this characteristic did not correlate with differences in microscopic morphology. In addition, the ΔPeSte12 mutants produced higher quantity of hydrogen peroxide than the wild type strain. Gene expression analysis revealed that PeSte12 was induced over time during apple infection compared to axenic growth, particularly from 2 dpi, reinforcing its role in virulence. Analysis of transcriptional abundance of several genes in ΔPeSte12 mutants showed that in most of the evaluated genes, PeSte12 seemed to act as a negative regulator during axenic growth, as most of them exhibited an increasing expression pattern along the time period evaluated. The highest expression values corresponded to detoxification, ATPase activity, protein folding and basic metabolism. Gene expression analysis during apple infection showed that 3 out of 9 analysed genes were up regulated; thus, PeSte12 seemed to exert a positive control to particular type of aldolase. These results demonstrate the PeSte12 transcription factor could play an important role in P. expansum's virulence and asexual reproduction.

Transcriptomic Response of Resistant (PI613981-Malus sieversii) and Susceptible ("Royal Gala") Genotypes of Apple to Blue Mold (Penicillium expansum) Infection.

Malus sieversii from Central Asia is a progenitor of the modern domesticated apple (Malus × domestica). Several accessions of M. sieversii are highly resistant to the postharvest pathogen Penicillium expansum. A previous study identified the qM-Pe3.1 QTL on LG3 for resistance to P. expansum in the mapping population GMAL4593, developed using the resistant accession, M. sieversii -PI613981, and the susceptible cultivar "Royal Gala" (RG) (M. domestica), as parents. The goal of the present study was to characterize the transcriptomic response of susceptible RG and resistant PI613981 apple fruit to wounding and inoculation with P. expansum using RNA-Seq. Transcriptomic analyses 0-48 h post inoculation suggest a higher basal level of resistance and a more rapid and intense defense response to wounding and wounding plus inoculation with P. expansum in M. sieversii -PI613981 than in RG. Functional analysis showed that ethylene-related genes and genes involved in "jasmonate" and "MYB-domain transcription factor family" were over-represented in the resistant genotype. It is suggested that the more rapid response in the resistant genotype (Malus sieversii-PI613981) plays a major role in the resistance response. At least twenty DEGs were mapped to the qM-Pe3.1 QTL (M × d v.1: 26,848,396-28,424,055) on LG3, and represent potential candidate genes responsible for the observed resistance QTL in M. sieversii-PI613981. RT-qPCR of several of these genes was used to validate the RNA-Seq data and to confirm their higher expression in MS0.

Identification and characterization of LysM effectors in Penicillium expansum.

P. expansum is regarded as one of the most important postharvest rots of apple fruit and is also of great concern to fruit processing industries. Elucidating the pathogenicity mechanism of this pathogen is of utmost importance for the development of effective and safe management strategies. Although, many studies on modification of the host environment by the pathogen were done, its interactions with fruit during the early stages of infection and the virulence factors that mediate pathogenicity have not been fully defined. Effectors carrying LysM domain have been identified in numerous pathogenic fungi and their role in the first stages of infection has been established. In this study, we identified 18 LysM genes in the P. expansum genome. Amino acid sequence analysis indicated that P. expansum LysM proteins belong to a clade of fungal-specific LysM. Eleven of the discovered LysM genes were found to have secretory pathway signal peptide, among them, 4 (PeLysM1 PeLysM2, PeLysM3 and PeLysM4) were found to be highly expressed during the infection and development of decay of apple fruit. Effect of targeted deletion of the four putative PeLysM effectors on the growth and pathogenicity was studied. Possible interactions of PeLysM with host proteins was investigated using the yeast-two-hybrid system.

LED Blue Light-induced changes in phenolics and ethylene in citrus fruit: Implication in elicited resistance against Penicillium digitatum infection.

The objective was to investigate whether LED Blue Light (LBL) induces changes in phenolics and ethylene production of sweet oranges, and whether they participate in LBL-elicited resistance against the most important postharvest pathogen (Penicillium digitatum) of citrus fruit. The expression of relevant genes of the phenylpropanoid and ethylene biosynthetic pathways during elicitation of resistance was also determined. Different LBL (wavelength 450nm) quantum fluxes were used within the 60-630µmolm-2s-1 range. The HPLC analysis showed that the most relevant increase in phenylpropanoids occurred in scoparone, which markedly increased 3days after exposing fruits to a very high quantum flux (630µmolm-2s-1) for 18h. However, phenylpropanoids, including scoparone, were not critical factors in LBL-induced resistance. The genes involved in ethylene biosynthesis were differentially regulated by LBL. Ethylene is not involved in elicited resistance, although high LBL levels increased ethylene production in only 1h.

Identification of Loci Affecting Accumulation of Secondary Metabolites in Tomato Fruit of a Solanum lycopersicum × Solanum chmielewskii Introgression Line Population.

Semi-polar metabolites such as flavonoids, phenolic acids, and alkaloids are very important health-related compounds in tomato. As a first step to identify genes responsible for the synthesis of semi-polar metabolites, quantitative trait loci (QTLs) that influence the semi-polar metabolite content in red-ripe tomato fruit were identified, by characterizing fruits of a population of introgression lines (ILs) derived from a cross between the cultivated tomato Solanum lycopersicum and the wild species Solanum chmielewskii. By analyzing fruits of plants grown at two different locations, we were able to identify robust metabolite QTLs for changes in phenylpropanoid glycoconjugation on chromosome 9, for accumulation of flavonol glycosides on chromosome 5, and for alkaloids on chromosome 7. To further characterize the QTLs we used a combination of genome sequencing, transcriptomics and targeted metabolomics to identify candidate key genes underlying the observed metabolic variation.