Botrytis cinerea infection accelerates ripening and cell wall disassembly to promote disease in tomato fruit.

Postharvest fungal pathogens benefit from the increased host susceptibility that occurs during fruit ripening. In unripe fruit, pathogens often remain quiescent and unable to cause disease until ripening begins, emerging at this point into destructive necrotrophic lifestyles that quickly result in fruit decay. Here, we demonstrate that one such pathogen, Botrytis cinerea, actively induces ripening processes to facilitate infections and promote disease in tomato (Solanum lycopersicum). Assessments of ripening progression revealed that B. cinerea accelerated external coloration, ethylene production, and softening in unripe fruit, while mRNA sequencing of inoculated unripe fruit confirmed the corresponding upregulation of host genes involved in ripening processes, such as ethylene biosynthesis and cell wall degradation. Furthermore, an enzyme-linked immunosorbent assay (ELISA)-based glycomics technique used to assess fruit cell wall polysaccharides revealed remarkable similarities in the cell wall polysaccharide changes caused by both infections of unripe fruit and ripening of healthy fruit, particularly in the increased accessibility of pectic polysaccharides. Virulence and additional ripening assessment experiments with B. cinerea knockout mutants showed that induction of ripening depends on the ability to infect the host and break down pectin. The B. cinerea double knockout Δbc polygalacturonase1 Δbc polygalacturonase2 lacking two critical pectin degrading enzymes was incapable of emerging from quiescence even long after the fruit had ripened at its own pace, suggesting that the failure to accelerate ripening severely inhibits fungal survival on unripe fruit. These findings demonstrate that active induction of ripening in unripe tomato fruit is an important infection strategy for B. cinerea.

Occurrence of Alternaria alternata and A. tenuissima causing black rot in cherry fruits (Prunus avium) in Central Chile.

During the harvest of 2020 and 2021, sweet cherry (Prunus avium) fruit showed a firm rot with irregular pale to dark brown lesions on the fruit surface, with green to light brown fungal growth resembling Alternaria-like infection (Simmons, 2007). Diseased cherries (n= 80 fruit) were collected at harvest in mature (over 10-year-old) commercial orchards of cherry tree varieties Lapins, Regina, Santina, Skeena, and Sweetheart planted in four localities of the regions O´Higgins (33°59´ S, 70°42´W; San Francisco de Mostazal and Graneros) and Maule (35°00'S, 71°23´W; Curicó and Sagrada Familia), Central Chile. The incidence of black rot was 1.9 and 3.2% in O´Higgins and Maule region, respectively, and it was increased to up to 5% during cold storage. The fruit collected previously, were transported to the lab, and surface disinfected in 75% ethanol for 15 s, and rinsed in sterile water. Internal pieces from the junction of diseased and healthy tissues of fruits were placed on potato dextrose agar (PDA, 2%) for 7 days at 20°C. Forty-two isolates of Alternaria-like (Simmons, 2007) were recovered consistently from pure cultures taking hyphal tips from 7 days old cultures. On PDA, 28 isolates (group A) were characterized by cottony, white-gray to green colonies and conidial chains (4 to 10 conidia) with secondary chains (1 to 5 conidia) branching on the conidiophore. Conidia were ovate to obclavate (mean 22.8 ± 5.1 x 8.8 ± 1.5 µm; n=40) with 3 to 7 transepta and 1 longisepta. The remaining 14 isolates (group B) were characterized by cottony, olive-green to olive-brown colonies following a ring pattern of growth and white margins, with conidial chains (4 to 14 conidia) and uncommon secondary chains (1 to 4 conidia). Conidia were obpyriform to ovate, light brown to brown with a cylindrical short beak at the tip (mean 24.7 ± 5.9 × 11.2 ± 1.3 µm; n=40) with 2 to 4 transepta, and 0 to 2 longisepta. Two representative isolates of group A (Sant-02-2020 and Bing-03-2020) and group B (Sant-26-2021 and Skeen-43-2021) were amplified for the Alternaria major allergen (Alt a1), plasma membrane ATPase (ATP), and calmodulin (Cal) loci following the protocols described by Hong et al. (2005) and Lawrence et al. (2013). A MegaBlast search of sequences of group A (GenBank nos. OR267293- OR267294, OR258001- OR258002, and OR267297- OR267298, for Alt a1, ATP, and Cal, respectively) showed 100% similarity to strains UCD10529 and UCD10539 of A. alternata, and group B (GenBank nos. OR267295- OR267296, OR258003- OR258004, and OR258005- OR258006, for Alt a1, ATP, and Cal, respectively) showed 100% similarity to strains EGS 34-015 and A30 of A. tenuissima. Combined phylogenetic analysis using MEGA X clustered isolates Sant-02-2020 and Bing-03-2020, and Sant-26-2021 and Skeen-43-2021 with ex-type of A. alternata and A. tenuissima, respectively. Pathogenicity tests were conducted using isolates of A. alternata (Sant-02-2020; Bing-03-2020) and A. tenuissima (Sant-26-2021; Skeen-43-2021). Detached ripe cherry fruit var. Sweetheart (n=40 fruits/isolate) and Regina (n=40 fruits/isolate) were surfaces disinfested (75% ethanol, 30 s), wounded in the middle with a sterile needle (2 mm in depth), and inoculated with 20 µL of conidial suspension (106 conidia/mL). An equal number of healthy cherries (n=40 fruits) treated with sterile water were used as controls. The experiment was repeated once. All inoculated fruit incubated for 7 days at 22°C, developed between 13 ± 2.7 to 23 ± 2.5 mm and 14.1 ± 1.1 to 19 ± 3.6 mm in lesion diameter for A. alternata and A. tenuissima isolates, respectively. Koch´s postulates were fulfilled by 100% reisolation of the causal pathogen from inoculated fruit, and molecular identification of A. alternata and A. tenuissima isolates. Previously, A. alternata has been described as causing rots on cherries in Chile (Acuña 2010), and China (Zhao and Liu, 2012; Ahmad et al., 2020). To our knowledge, this is the first occurrence of cherry black rot caused by A. alternata and A. tenuissima in Central Chile. Epidemiological studies are necessary to develop integrated management of cherry black rot in Central Chile.

Host susceptibility factors render ripe tomato fruit vulnerable to fungal disease despite active immune responses.

The increased susceptibility of ripe fruit to fungal pathogens poses a substantial threat to crop production and marketability. Here, we coupled transcriptomic analyses with mutant studies to uncover critical processes associated with defense and susceptibility in tomato (Solanum lycopersicum) fruit. Using unripe and ripe fruit inoculated with three fungal pathogens, we identified common pathogen responses reliant on chitinases, WRKY transcription factors, and reactive oxygen species detoxification. We established that the magnitude and diversity of defense responses do not significantly impact the interaction outcome, as susceptible ripe fruit mounted a strong immune response to pathogen infection. Then, to distinguish features of ripening that may be responsible for susceptibility, we utilized non-ripening tomato mutants that displayed different susceptibility patterns to fungal infection. Based on transcriptional and hormone profiling, susceptible tomato genotypes had losses in the maintenance of cellular redox homeostasis, while jasmonic acid accumulation and signaling coincided with defense activation in resistant fruit. We identified and validated a susceptibility factor, pectate lyase (PL). CRISPR-based knockouts of PL, but not polygalacturonase (PG2a), reduced susceptibility of ripe fruit by >50%. This study suggests that targeting specific genes that promote susceptibility is a viable strategy to improve the resistance of tomato fruit against fungal disease.

Characterization of CRISPR Mutants Targeting Genes Modulating Pectin Degradation in Ripening Tomato.

Tomato (Solanum lycopersicum) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals, and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodeling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin-degrading enzymes are involved in cell wall remodeling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin-degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a), and ß-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL, PG2a, and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to ß-1,4-galactan, and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a, and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines.

The genomic diversification of grapevine clones.

BACKGROUND: Vegetatively propagated clones accumulate somatic mutations. The purpose of this study was to better appreciate clone diversity and involved defining the nature of somatic mutations throughout the genome. Fifteen Zinfandel winegrape clone genomes were sequenced and compared to one another using a highly contiguous genome reference produced from one of the clones, Zinfandel 03. RESULTS: Though most heterozygous variants were shared, somatic mutations accumulated in individual and subsets of clones. Overall, heterozygous mutations were most frequent in intergenic space and more frequent in introns than exons. A significantly larger percentage of CpG, CHG, and CHH sites in repetitive intergenic space experienced transition mutations than in genic and non-repetitive intergenic spaces, likely because of higher levels of methylation in the region and because methylated cytosines often spontaneously deaminate. Of the minority of mutations that occurred in exons, larger proportions of these were putatively deleterious when they occurred in relatively few clones. CONCLUSIONS: These data support three major conclusions. First, repetitive intergenic space is a major driver of clone genome diversification. Second, clones accumulate putatively deleterious mutations. Third, the data suggest selection against deleterious variants in coding regions or some mechanism by which mutations are less frequent in coding than noncoding regions of the genome.

Red blotch disease alters grape berry development and metabolism by interfering with the transcriptional and hormonal regulation of ripening.

Grapevine red blotch-associated virus (GRBaV) is a major threat to the wine industry in the USA. GRBaV infections (aka red blotch disease) compromise crop yield and berry chemical composition, affecting the flavor and aroma properties of must and wine. In this study, we combined genome-wide transcriptional profiling with targeted metabolite analyses and biochemical assays to characterize the impact of the disease on red-skinned berry ripening and metabolism. Using naturally infected berries collected from two vineyards, we were able to identify consistent berry responses to GRBaV across different environmental and cultural conditions. Specific alterations of both primary and secondary metabolism occurred in GRBaV-infected berries during ripening. Notably, GRBaV infections of post-véraison berries resulted in the induction of primary metabolic pathways normally associated with early berry development (e.g. thylakoid electron transfer and the Calvin cycle), while inhibiting ripening-associated pathways, such as a reduced metabolic flux in the central and peripheral phenylpropanoid pathways. We show that this metabolic reprogramming correlates with perturbations at multiple regulatory levels of berry development. Red blotch caused the abnormal expression of transcription factors (e.g. NACs, MYBs, and AP2-ERFs) and elements of the post-transcriptional machinery that function during red-skinned berry ripening. Abscisic acid, ethylene, and auxin pathways, which control both the initiation of ripening and stress responses, were also compromised. We conclude that GRBaV infections disrupt normal berry development and stress responses by altering transcription factors and hormone networks, which result in the inhibition of ripening pathways involved in the generation of color, flavor, and aroma compounds.

Developmental and Metabolic Plasticity of White-Skinned Grape Berries in Response to Botrytis cinerea during Noble Rot.

Noble rot results from exceptional infections of ripe grape (Vitis vinifera) berries by Botrytis cinerea. Unlike bunch rot, noble rot promotes favorable changes in grape berries and the accumulation of secondary metabolites that enhance wine grape composition. Noble rot-infected berries of cv Sémillon, a white-skinned variety, were collected over 3 years from a commercial vineyard at the same time that fruit were harvested for botrytized wine production. Using an integrated transcriptomics and metabolomics approach, we demonstrate that noble rot alters the metabolism of cv Sémillon berries by inducing biotic and abiotic stress responses as well as ripening processes. During noble rot, B. cinerea induced the expression of key regulators of ripening-associated pathways, some of which are distinctive to the normal ripening of red-skinned cultivars. Enhancement of phenylpropanoid metabolism, characterized by a restricted flux in white-skinned berries, was a common outcome of noble rot and red-skinned berry ripening. Transcript and metabolite analyses together with enzymatic assays determined that the biosynthesis of anthocyanins is a consistent hallmark of noble rot in cv Sémillon berries. The biosynthesis of terpenes and fatty acid aroma precursors also increased during noble rot. We finally characterized the impact of noble rot in botrytized wines. Altogether, the results of this work demonstrated that noble rot causes a major reprogramming of berry development and metabolism. This desirable interaction between a fruit and a fungus stimulates pathways otherwise inactive in white-skinned berries, leading to a greater accumulation of compounds involved in the unique flavor and aroma of botrytized wines.

Distinctive expansion of gene families associated with plant cell wall degradation, secondary metabolism, and nutrient uptake in the genomes of grapevine trunk pathogens.

BACKGROUND: Trunk diseases threaten the longevity and productivity of grapevines in all viticulture production systems. They are caused by distantly-related fungi that form chronic wood infections. Variation in wood-decay abilities and production of phytotoxic compounds are thought to contribute to their unique disease symptoms. We recently released the draft sequences of Eutypa lata, Neofusicoccum parvum and Togninia minima, causal agents of Eutypa dieback, Botryosphaeria dieback and Esca, respectively. In this work, we first expanded genomic resources to three important trunk pathogens, Diaporthe ampelina, Diplodia seriata, and Phaeomoniella chlamydospora, causal agents of Phomopsis dieback, Botryosphaeria dieback, and Esca, respectively. Then we integrated all currently-available information into a genome-wide comparative study to identify gene families potentially associated with host colonization and disease development. RESULTS: The integration of RNA-seq, comparative and ab initio approaches improved the protein-coding gene prediction in T. minima, whereas shotgun sequencing yielded nearly complete genome drafts of Dia. ampelina, Dip. seriata, and P. chlamydospora. The predicted proteomes of all sequenced trunk pathogens were annotated with a focus on functions likely associated with pathogenesis and virulence, namely (i) wood degradation, (ii) nutrient uptake, and (iii) toxin production. Specific patterns of gene family expansion were described using Computational Analysis of gene Family Evolution, which revealed lineage-specific evolution of distinct mechanisms of virulence, such as specific cell wall oxidative functions and secondary metabolic pathways in N. parvum, Dia. ampelina, and E. lata. Phylogenetically-informed principal component analysis revealed more similar repertoires of expanded functions among species that cause similar symptoms, which in some cases did not reflect phylogenetic relationships, thereby suggesting patterns of convergent evolution. CONCLUSIONS: This study describes the repertoires of putative virulence functions in the genomes of ubiquitous grapevine trunk pathogens. Gene families with significantly faster rates of gene gain can now provide a basis for further studies of in planta gene expression, diversity by genome re-sequencing, and targeted reverse genetic approaches. The functional validation of potential virulence factors will lead to a more comprehensive understanding of the mechanisms of pathogenesis and virulence, which ultimately will enable the development of accurate diagnostic tools and effective disease management.

Non-wounding contact-based Inoculation of fruits with fungal pathogens in postharvest.

BACKGROUND: Fungal pathogens significantly impact the quality of fruits and vegetables at different stages of the supply chain, leading to substantial food losses. Understanding how these persistent fungal infections occur and progress in postharvest conditions is essential to developing effective control strategies. RESULTS: In this study, we developed a reliable and consistent inoculation protocol to simulate disease spread from infected fruits to adjacent healthy fruits during postharvest storage. We tested different combinations of relevant fruit commodities, including oranges, tomatoes, and apples, against impactful postharvest pathogens such as Penicillium digitatum, Penicillium italicum, Botrytis cinerea, and Penicillium expansum. We assessed the efficacy of this protocol using fruits treated with various postharvest methods and multiple isolates for each pathogen. We optimized the source of infected tissue and incubation conditions for each fruit-pathogen combination. Disease incidence and severity were quantitatively evaluated to study infection success and progression. At the final evaluation point, 80% or higher disease incidence rates were observed in all trials except for the fungicide-treated oranges inoculated with fungicide-susceptible Penicillium spp. isolates. Although disease incidence was lower in that particular scenario, it is noteworthy that the pathogen was still able to establish itself under unfavorable conditions, indicating the robustness of our methodology. Finally, we used multispectral imaging to detect early P. digitatum infections in oranges before the disease became visible to the naked eye but after the pathogen was established. CONCLUSIONS: We developed a non-invasive inoculation strategy that can be used to recreate infections caused by contact or nesting in postharvest. The observed high disease incidence and severity values across fruit commodities and fungal pathogens demonstrate the robustness, efficacy, and reproducibility of the developed methodology. The protocol has the potential to be tailored for other pathosystems. Additionally, this approach can facilitate the study of fruit-pathogen interactions and the assessment of innovative control strategies.

Targeting ripening regulators to develop fruit with high quality and extended shelf life.

Fruit quality directly impacts fruit marketability and consumer acceptance. Breeders have focused on fruit quality traits to extend shelf life, primarily through fruit texture, but, in some cases, have neglected other qualities such as flavor and nutrition. In recent years, integrative biotechnology and consumer-minded approaches have surfaced, aiding in the development of flavorful, long-lasting fruit. Here, we discussed how specific transcription factors and hormones involved in fruit ripening can be targeted to generate high-quality fruit through traditional breeding and bioengineering. We highlight regulators that can be used to generate novel-colored fruit or biofortify fresh produce with health-promoting nutrients, such as vitamin C. Overall, we argue that addressing grower and industry needs must be balanced with consumer-based traits.

Interactive effect of branch source-sink ratio and leaf aging on photosynthesis in pistachio.

Tree source-sink ratio has a predominant and complex impact on tree performance and can affect multiple physiological processes including vegetative and reproductive growth, water and nutrient use, photosynthesis, and productivity. In this study, we manipulated the branch level source-sink ratio by reduction of photosynthetic activity (partial branch defoliation) or thinning branch fruit load early in the growing season (after fruit set) in pistachio (Pistacia vera) trees. We then characterized the leaf photosynthetic light response curves through leaf aging. In addition, we determined changes in leaf non-structural carbohydrates (NSC) and nitrogen (N) concentrations. In leaves with high source-sink ratios, there was a gradual decrease in maximum net photosynthetic rate (ANmax) over the growing season, while in branches with low source-sink ratios, there was a sharp decline in ANmax in the first two weeks of August. Branches with high-sink showed an up-regulation (increase) in photosynthesis toward the end of July (at 1,500 growing degree days) during the period of rapid kernel growth rate and increased sink strength, with ANmax being about 7 µmol m-1 s-1 higher than in branches with low-sink. In August, low source-sink ratios precipitated leaf senescence, resulting in a drastic ANmax decline, from 25 to 8 µmol m-1 s-1 (70% drop in two weeks). This reduction was associated with the accumulation of NSC in the leaves from 20 to 30 mg g-1. The mechanisms of ANmax reduction differ between the two treatments. Lower photosynthetic rates of 8-10 µmol m-1 s-1 late in the season were associated with lower N levels in high-sink branches, suggesting N remobilization to the kernels. Lower photosynthesis late in the season was associated with lower respiration rates in low-source branches, indicating prioritization of assimilates to storage. These results can facilitate the adaptation of management practices to tree crop load changes in alternate bearing species.

Transcriptional profiling of the terpenoid biosynthesis pathway and in vitro tests reveal putative roles of linalool and farnesal in nectarine resistance against brown rot.

The most devastating fungal disease of peaches and nectarines is brown rot, caused by Monilinia spp. Among the many plant responses against biotic stress, plant terpenoids play essential protective functions, including antioxidant activities and inhibition of pathogen growth. Herein, we aimed to characterize the expression of terpenoid biosynthetic genes in fruit tissues that presented different susceptibility to brown rot. For that, we performed artificial inoculations with Monilinia laxa at two developmental stages (immature and mature fruit) of two nectarine cultivars ('Venus' -mid-early season cultivar - and 'Albared' -late season cultivar-) and in vitro tests of the key compounds observed in the transcriptional results. All fruit were susceptible to M. laxa except for immature 'Venus' nectarines. In response to the pathogen, the mevalonic acid (MVA) pathway of the 'Venus' cultivar was highly induced in both stages rather than the methylerythritol phosphate (MEP) pathway, being the expression of some MEP-related biosynthetic genes [e.g., PROTEIN FARNESYLTRANSFERASE (PpPFT), and 3S-LINALOOL SYNTHASE (PpLIS)] different between stages. In 'Albared', both stages presented similar responses to M. laxa for both pathways. Comparisons between cultivars showed that HYDROXYMETHYLGLUTARYL-CoA REDUCTASE (PpHMGR1) expression levels were common in susceptible tissues. Within all the terpenoid biosynthetic pathway, linalool- and farnesal-related pathways stood out for being upregulated only in resistant tissues, which suggest their role in mediating the resistance to M. laxa. The in vitro antifungal activity of linalool and farnesol (precursor of farnesal) revealed fungicidal and fungistatic activities against M. laxa, respectively, depending on the concentration tested. Understanding the different responses between resistant and susceptible tissues could be further considered for breeding or developing new strategies to control brown rot in stone fruit.

A pear S1-bZIP transcription factor PpbZIP44 modulates carbohydrate metabolism, amino acid, and flavonoid accumulation in fruits.

Fruit quality is defined by attributes that give value to a commodity. Flavor, texture, nutrition, and shelf life are key quality traits that ensure market value and consumer acceptance. In pear fruit, soluble sugars, organic acids, amino acids, and total flavonoids contribute to flavor and overall quality. Transcription factors (TFs) regulate the accumulation of these metabolites during development or in response to the environment. Here, we report a novel TF, PpbZIP44, as a positive regulator of primary and secondary metabolism in pear fruit. Analysis of the transient overexpression or RNAi-transformed pear fruits and stable transgenic tomato fruits under the control of the fruit-specific E8 promoter demonstrated that PpZIP44 substantially affected the contents of soluble sugar, organic acids, amino acids, and flavonoids. In E8::PpbZIP44 tomato fruit, genes involved in carbohydrate metabolism, amino acid, and flavonoids biosynthesis were significantly induced. Furthermore, in PpbZIP44 overexpression or antisense pear fruits, the expression of genes in the related pathways was significantly impacted. PpbZIP44 directly interacted with the promoter of PpSDH9 and PpProDH1 to induce their expression, thereby depleting sorbitol and proline, decreasing citrate and malate, and enhancing fructose contents. PpbZIP44 also directly bound to the PpADT and PpF3H promoters, which led to the carbon flux toward phenylalanine metabolites and enhanced phenylalanine and flavonoid contents. These findings demonstrate that PpbZIP44 mediates multimetabolism reprogramming by regulating the gene expression related to fruit quality compounds.

Coordinated transcriptional regulation of the carotenoid biosynthesis contributes to fruit lycopene content in high-lycopene tomato genotypes.

Lycopene content in tomato fruit is largely under genetic control and varies greatly among genotypes. Continued improvement of lycopene content in elite varieties with conventional breeding has become challenging, in part because little is known about the underlying molecular mechanisms in high-lycopene tomatoes (HLYs). We collected 42 HLYs with different genetic backgrounds worldwide. High-performance liquid chromatography (HPLC) analysis revealed lycopene contents differed among the positive control wild tomato Solanum pimpinellifolium, HLYs, the normal lycopene cultivar "Moneymaker", and the non-lycopene cultivar NC 1Y at the pink and red ripe stages. Real-time RT-PCR analysis of expression of the 25 carotenoid biosynthesis pathway genes of each genotype showed a significantly higher expression in nine upstream genes (GGPPS1, GGPPS2, GGPPS3, TPT1, SSU II, PSY2, ZDS, CrtISO and CrtISO-L1 but not the well-studied PSY1, PDS and Z-ISO) at the breaker and/or red ripe stages in HLYs compared to Moneymaker, indicating a higher metabolic flux flow into carotenoid biosynthesis pathway in HLYs. Further conversion of lycopene to carotenes may be prevented via the two downstream genes (ß-LCY2 and ε-LCY), which had low-abundance transcripts at either or both stages. Additionally, the significantly higher expression of four downstream genes (BCH1, ZEP, VDE, and CYP97C11) at either or both ripeness stages leads to significantly lower fruit lycopene content in HLYs than in the wild tomato. This is the first systematic investigation of the role of the complete pathway genes in regulating fruit lycopene biosynthesis across many HLYs, and enables tomato breeding and gene editing for increased fruit lycopene content.

Genomic prediction of strawberry resistance to postharvest fruit decay caused by the fungal pathogen Botrytis cinerea.

Gray mold, a disease of strawberry (Fragaria × ananassa) caused by the ubiquitous necrotroph Botrytis cinerea, renders fruit unmarketable and causes economic losses in the postharvest supply chain. To explore the feasibility of selecting for increased resistance to gray mold, we undertook genetic and genomic prediction studies in strawberry populations segregating for fruit quality and shelf life traits hypothesized to pleiotropically affect susceptibility. As predicted, resistance to gray mold was heritable but quantitative and genetically complex. While every individual was susceptible, the speed of symptom progression and severity differed. Narrow-sense heritability ranged from 0.38 to 0.71 for lesion diameter (LD) and 0.39 to 0.44 for speed of emergence of external mycelium (EM). Even though significant additive genetic variation was observed for LD and EM, the phenotypic ranges were comparatively narrow and genome-wide analyses did not identify any large-effect loci. Genomic selection (GS) accuracy ranged from 0.28 to 0.59 for LD and 0.37 to 0.47 for EM. Additive genetic correlations between fruit quality and gray mold resistance traits were consistent with prevailing hypotheses: LD decreased as titratable acidity increased, whereas EM increased as soluble solid content decreased and firmness increased. We concluded that phenotypic and GS could be effective for reducing LD and increasing EM, especially in long shelf life populations, but that a significant fraction of the genetic variation for resistance to gray mold was caused by the pleiotropic effects of fruit quality traits that differ among market and shelf life classes.

Single and Double Mutations in Tomato Ripening Transcription Factors Have Distinct Effects on Fruit Development and Quality Traits.

Spontaneous mutations associated with the tomato transcription factors COLORLESS NON-RIPENING (SPL-CNR), NON-RIPENING (NAC-NOR), and RIPENING-INHIBITOR (MADS-RIN) result in fruit that do not undergo the normal hallmarks of ripening but are phenotypically distinguishable. Here, we expanded knowledge of the physiological, molecular, and genetic impacts of the ripening mutations on fruit development beyond ripening. We demonstrated through phenotypic and transcriptome analyses that Cnr fruit exhibit a broad range of developmental defects before the onset of fruit ripening, but fruit still undergo some ripening changes similar to wild type. Thus, Cnr should be considered as a fruit developmental mutant and not just a ripening mutant. Additionally, we showed that some ripening processes occur during senescence in the nor and rin mutant fruit, indicating that while some ripening processes are inhibited in these mutants, others are merely delayed. Through gene expression analysis and direct measurement of hormones, we found that Cnr, nor, and rin have alterations in the metabolism and signaling of plant hormones. Cnr mutants produce more than basal levels of ethylene, while nor and rin accumulate high concentrations of abscisic acid. To determine genetic interactions between the mutations, we created for the first time homozygous double mutants. Phenotypic analyses of the double ripening mutants revealed that Cnr has a strong influence on fruit traits and that combining nor and rin leads to an intermediate ripening mutant phenotype. However, we found that the genetic interactions between the mutations are more complex than anticipated, as the Cnr/nor double mutant fruit has a Cnr phenotype but displayed inhibition of ripening-related gene expression just like nor fruit. Our reevaluation of the Cnr, nor, and rin mutants provides new insights into the utilization of the mutants for studying fruit development and their implications in breeding for tomato fruit quality.

Depicting the battle between nectarine and Monilinia laxa: the fruit developmental stage dictates the effectiveness of the host defenses and the pathogen's infection strategies.

Infections by the fungus Monilinia laxa, the main cause of brown rot in Europe, result in considerable losses of stone fruit. Herein, we present a comprehensive transcriptomic approach to unravel strategies deployed by nectarine fruit and M. laxa during their interaction. We used M. laxa-inoculated immature and mature fruit, which was resistant and susceptible to brown rot, respectively, to perform a dual RNA-Seq analysis. In immature fruit, host responses, pathogen biomass, and pathogen transcriptional activity peaked at 14-24 h post inoculation (hpi), at which point M. laxa appeared to switch its transcriptional response to either quiescence or death. Mature fruit experienced an exponential increase in host and pathogen activity beginning at 6 hpi. Functional analyses in both host and pathogen highlighted differences in stage-dependent strategies. For example, in immature fruit, M. laxa unsuccessfully employed carbohydrate-active enzymes (CAZymes) for penetration, which the fruit was able to combat with tightly regulated hormone responses and an oxidative burst that challenged the pathogen's survival at later time points. In contrast, in mature fruit, M. laxa was more dependent on proteolytic effectors than CAZymes, and was able to invest in filamentous growth early during the interaction. Hormone analyses of mature fruit infected with M. laxa indicated that, while jasmonic acid activity was likely useful for defense, high ethylene activity may have promoted susceptibility through the induction of ripening processes. Lastly, we identified M. laxa genes that were highly induced in both quiescent and active infections and may serve as targets for control of brown rot.

Grey mould of strawberry, a devastating disease caused by the ubiquitous necrotrophic fungal pathogen Botrytis cinerea.

The fungal pathogen Botrytis cinerea causes grey mould, a commercially damaging disease of strawberry. This pathogen affects fruit in the field, storage, transport and market. The presence of grey mould is the most common reason for fruit rejection by growers, shippers and consumers, leading to significant economic losses. Here, we review the biology and epidemiology of the pathogen, mechanisms of infection and the genetics of host plant resistance. The development of grey mould is affected by environmental and genetic factors; however, little is known about how B. cinerea and strawberry interact at the molecular level. Despite intensive efforts, breeding strawberry for resistance to grey mould has not been successful, and the mechanisms underlying tolerance to B. cinerea are poorly understood and under-investigated. Current control strategies against grey mould include pre- and postharvest fungicides, yet they are generally ineffective and expensive. In this review, we examine available research on horticultural management, chemical and biological control of the pathogen in the field and postharvest storage, and discuss their relevance for integrative disease management. Additionally, we identify and propose approaches for increasing resistance to B. cinerea in strawberry by tapping into natural genetic variation and manipulating host factors via genetic engineering and genome editing.

Iso-Seq Allows Genome-Independent Transcriptome Profiling of Grape Berry Development.

Transcriptomics has been widely applied to study grape berry development. With few exceptions, transcriptomic studies in grape are performed using the available genome sequence, PN40024, as reference. However, differences in gene content among grape accessions, which contribute to phenotypic differences among cultivars, suggest that a single reference genome does not represent the species' entire gene space. Though whole genome assembly and annotation can reveal the relatively unique or "private" gene space of any particular cultivar, transcriptome reconstruction is a more rapid, less costly, and less computationally intensive strategy to accomplish the same goal. In this study, we used single molecule-real time sequencing (SMRT) to sequence full-length cDNA (Iso-Seq) and reconstruct the transcriptome of Cabernet Sauvignon berries during berry ripening. In addition, short reads from ripening berries were used to error-correct low-expression isoforms and to profile isoform expression. By comparing the annotated gene space of Cabernet Sauvignon to other grape cultivars, we demonstrate that the transcriptome reference built with Iso-Seq data represents most of the expressed genes in the grape berries and includes 1,501 cultivar-specific genes. Iso-Seq produced transcriptome profiles similar to those obtained after mapping on a complete genome reference. Together, these results justify the application of Iso-Seq to identify cultivar-specific genes and build a comprehensive reference for transcriptional profiling that circumvents the necessity of a genome reference with its associated costs and computational weight.

Infection Strategies Deployed by Botrytis cinerea, Fusarium acuminatum, and Rhizopus stolonifer as a Function of Tomato Fruit Ripening Stage.

Worldwide, 20-25% of all harvested fruit and vegetables are lost annually in the field and throughout the postharvest supply chain due to rotting by fungal pathogens. Most postharvest pathogens exhibit necrotrophic or saprotrophic lifestyles, resulting in decomposition of the host tissues and loss of marketable commodities. Necrotrophic fungi can readily infect ripe fruit leading to the rapid establishment of disease symptoms. However, these pathogens generally fail to infect unripe fruit or remain quiescent until host conditions stimulate a successful infection. Previous research on infections of fruit has mainly been focused on the host's genetic and physicochemical factors that inhibit or promote disease. Here, we investigated if fruit pathogens can modify their own infection strategies in response to the ripening stage of the host. To test this hypothesis, we profiled global gene expression of three fungal pathogens that display necrotrophic behavior-Botrytis cinerea, Fusarium acuminatum, and Rhizopus stolonifer-during interactions with unripe and ripe tomato fruit. We assembled and functionally annotated the transcriptomes of F. acuminatum and R. stolonifer as no genomic resources were available. Then, we conducted differential gene expression analysis to compare each pathogen during inoculations versus in vitro conditions. Through characterizing patterns of overrepresented pathogenicity and virulence functions (e.g., phytotoxin production, cell wall degradation, and proteolysis) among the differentially expressed genes, we were able to determine shared strategies among the three fungi during infections of compatible (ripe) and incompatible (unripe) fruit tissues. Though each pathogen's strategy differed in the details, interactions with unripe fruit were commonly characterized by an emphasis on the degradation of cell wall components, particularly pectin, while colonization of ripe fruit featured more heavily redox processes, proteolysis, metabolism of simple sugars, and chitin biosynthesis. Furthermore, we determined that the three fungi were unable to infect fruit from the non-ripening (nor) tomato mutant, confirming that to cause disease, these pathogens require the host tissues to undergo specific ripening processes. By enabling a better understanding of fungal necrotrophic infection strategies, we move closer to generating accurate models of fruit diseases and the development of early detection tools and effective management strategies.