Although we have not characterized the type of inoculum that is spreading from source to target fruit, our observations that source fruits or tissue sections without visible mycelium or sporulation were more effective in contact inoculation, we hypothesize that the disease is spread by fungal hyphae that are moving from infected tissues to healthy ones in search of nutrients. Ultimately, this protocol offers an effective and robust method for studying fruit-pathogen interactions and can be used to test the efficacy of post harvest treatments against persistent post harvest pathogens.Fungi are important plant pathogens that cause large economic losses due to their ability to inflict diseases such as rot, rust, and wilt in various plant organs both preharvest and post harvest . Biotrophic fungi feed on living cells and suppress the host immune system by secreting effector proteins . In contrast, necrotrophic fungi feed on dead host cells and cause necrosis by secreting toxins and cell wall degrading enzymes , among other virulence factors . Due to their ability to feed on dead host tissue, necrotrophic fungi are also sometimes grouped into the less defined group of saprotrophic fungi, which includes many fungi that do not actively kill host cells . Additionally, drainage planter pot hemibiotrophs are pathogens that start their infection cycle as biotrophs and end as necrotrophs . Biotrophic infection mechanisms are well-studied, whereas those of necrotrophic fungi are less understood.
The lower scientific interest in necrotrophic infection mechanisms may be due to their perceived lack of specificity. The brute force strategy of secreting toxins and CWDEs as well as the broad host range of many necrotrophic fungi is often interpreted as indiscriminate killing of host cells without the requirement for host-pathogen compatibility . However, the reality of necrotrophic infections is multifaceted, as they involve several features initially believed to be unique to biotrophs, e.g., the suppression of the host immune system or symptomless endophytic growth . The relevance of host-pathogen compatibility in necrotrophic infections is also highlighted by the fact that necrotrophic fungi can readily infect ripe fruit but fail to infect unripe fruit or remain quiescent until host and environmental conditions stimulate a successful infection . To develop a better understanding of how fungi attempt to establish infections in fruit, we studied three impactful pathogens with broad host range: Botrytis cinerea, Fusarium acuminatum, and Rhizopus stolonifer. B. cinerea is the causal agent of gray mold, an economically devastating disease, and serves as a model species for plant-necrotroph interactions . In compatible hosts, such as ripe fruit, B. cinerea produces toxins, CWDEs, reactive oxygen species , and other virulence factors to induce rapid death and decay of the plant tissues .
In incompatible hosts, such as unripe fruit, B. cinerea establishes quiescent infections while suppressing the host immune system and promoting susceptibility in the host . B. cinerea has been shown to activate fruit ripening processes, including changes in plant hormone biosynthesis and signaling and induction of host CWDEs involved in fruit softening, all of which seem to favor fungal growth and colonization . Even though B. cinerea infection strategies have been studied in various pathosystems , it is mostly unknown whether F. acuminatum and R. stolonifer, two understudied fungal pathogens, implement similar mechanisms when interacting with compatible and incompatible hosts. F. acuminatum has been reported to infect roots and fruit . Within the Fusarium genus, F. acuminatum is among the most toxic species as it produces strong mycotoxins, such as trichothecene and fumonisins, to kill host cells and induce tissue necrosis . R. stolonifer causes rotting of fruit and other fresh products, mainly by secreting CWDEs, and is considered to be one of the most destructive post harvest pathogens due to its extremely fast growth rate . We leveraged the fact that tomato fruit display an increase in susceptibility to necrotrophic fungal infection as a result of ripening to develop a system for studying compatible and incompatible host-pathogen interactions. The transition from unripe to ripe fruit results in a markedly different physicochemical environment for colonization. In comparison to unripe fruit, ripe fruit have higher levels of total soluble solids, greater titratable acidity , lower firmness, and a different composition of secondary metabolites and volatiles .
In light of this, we anticipated that these pathogens would exhibit specific patterns of gene expression based on the fruit ripening stage and that the functions of these genes would reflect important strategies for interaction with the different host environments. First, we evaluated the incidence and progression of fungal infections caused by B. cinerea, F. acuminatum, and R. stolonifer when inoculated in tomato fruit. Then, to determine if the pathogens adapted their infection strategies as a function of the host developmental stage, we analyzed the transcriptomes of each fungus at two points post-inoculation in unripe and ripe tomato fruit and compared these against their transcriptomes when grown under in vitro conditions. This approach allowed us to identify specific pathogenicity and virulence factors, e.g., CWDEs and toxin biosynthetic genes, that are differentially or commonly deployed by the pathogens in each host tissue. As necrotrophic infection strategies may be evolutionarily conserved as well as highly specific, we used the transcriptomic data toexamine virulence functions among the three fungi and identified similarities in the adaptations of the pathogens to the different environments of ripe and unripe fruit. Finally, to further validate necrotrophic strategies dependent on the ripening stage of the fruit host, we evaluated the pathogenicity of the three fungal pathogens in fruit in a non-ripening tomato mutant. Overall, the approach followed in this study provides an initial platform to perform comparative transcriptomics among three fungi that cause economically relevant fruit diseases and sheds light into how pathogens with necrotrophic lifestyles adapt their infection mechanisms during compatible and incompatible interactions.Tomato fruit from the cultivar Ailsa Craig and the isogenic mutant non-ripening were used in this study. Plants were grown under field conditions in Davis, CA, United States, during the 2017 season. Mature green fruit were harvested 31 days post-anthesis and red ripe fruit at 42 dpa. The fungal pathogens studied were B. cinerea strain B05.10, an isolate of R. stolonifer, and an isolate of F. acuminatum. The isolates of R. stolonifer and F. acuminatum were obtained from post harvest infections of fresh produce and identified using morphological and sequencing methods. All fungi were grown on 1% potato dextrose agar plates at room temperature until sporulation. Spore suspensions were prepared in 0.01% TweenR 20 . Fungi from axenic in vitro cultures were grown on 1% PDA plates at RT, and mycelium for RNA extraction was harvested before the fungi reached the sporulation stage.Tomato fruit from AC and nor were collected at MG and RR or RR-like stage. Selected AC MG fruit were green, firm, and had soluble solids content of 5.24 ± 0.44 g sucrose/100 g solution and TA of 5.20 ± 1.24%. AC RR fruit were bright red, pliable when squeezed, and had SSC of 6.27 ± 0.42 g sucrose/100 g solution and a TA of 3.47 ± 0.26%. nor MG fruit were similar to AC MG fruit, and nor RR-like fruit were selected that were green in color slightly soft at the blossom end. Fruit were sterilized in 0.6% sodium hypochlorite, wounded four to six times on the blossom end with a sterile pipette tip and inoculated with 10 µl per wound using a 500 spores/µl suspension for B. cinerea, a 30 spores/µl suspension of R. stolonifer, and a 1,000 spores/µl suspension of F. acuminatum.
The differences in fungal spore concentration were adjusted to ensure uniform and comparable development of lesions in tomato fruit. In the case of R. stolonifer inoculations of MG fruit, plant pot with drainage we also tested a concentration of 1,000 spores/µl but no differences in fruit responses or fungal growth between this concentration and 30 spores/µl were observed. Inoculated fruit were incubated at RT in high humidity chambers. For mock inoculations, the same procedure was followed but without the addition of the inoculum. The pericarp and epidermis of the blossom end were collected at 1 and 3 days post-inoculation , immediately frozen in liquid nitrogen, and stored at −80◦C until use. One biological replicate consisted on average of eight fruit, and five biological replicates per treatment were obtained.Tomato fruit tissues were ground using a RetschR Mixer Mill MM 400 and RNA was extracted from 1 g of fine-powdered tissue according to the procedure described in Blanco-Ulate et al. . Fungal RNA from the in vitro cultures was extracted using TRIzol and purified using the Quick-RNA MiniPrep Kit following the procedure described in MoralesCruz et al. . The RNA concentration and purity were assessed with the Qubit 3 and the NanoDrop One Spectrophotometer , respectively. Barcoded cDNA libraries were prepared using the Illumina TruSeq RNA Sample Preparation Kit v2 . Quality control of the cDNA libraries was performed with the High Sensitivity DNA Analysis Kit in the Agilent 2100 Bioanalyzer . 50-bp single-end libraries were sequenced on the Illumina HiSeq 4000 platform in the DNA Technologies Core of the UC Davis Genome Center. In total, 18 libraries were sequenced for B. cinerea , 17 libraries were sequenced for F. acuminatum , and 17 libraries for R. stolonifer .To determine if F. acuminatum and R. stolonifer show similar patterns of infections in tomato fruit as B. cinerea , we did side-by-side inoculations of fruit at two developmental stages: unripe and ripe . As displayed in Figure 1A, we confirmed that all fungi were unable to cause rotting in MG fruit but aggressively colonized RR fruit. These results were further validated by quantifying fungal biomass based on relative expression of fungal reference genes via qRT-PCR . At 3 dpi, RR fruit inoculated with B. cinerea and F. acuminatum showed water-soaked lesions of approximately 15 mm covered by dense mycelia, whereas RR fruit inoculated with R. stolonifer were almost decomposed and entirely covered by mycelia. Although no lesions were observed in MG fruit when inoculated with any of the pathogens, some differences in fungal growth and tomato responses were observed. Inoculations with B. cinerea and R. stolonifer did not show any visible mycelia, whereas F. acuminatum inoculations showed limited hyphal growth without disease symptoms. All three fungi induced a necrotic ring surrounding the inoculation sites during the incompatible interaction with MG fruit, yet F. acuminatum inoculations caused dark and wide rings while fruit infected with R. stolonifer developed a weaker response. Because we were not able to visually detect any hyphal growth of B. cinerea and R. stolonifer inMG fruit, we used a microscope to observe whether the spores germinated in the inoculated wounds. At 1 dpi, B. cinerea spores were mainly ungerminated or in the process of germination . By contrast, F. acuminatum and R. stolonifer already showed active hyphal growth, indicating that spores of these fungi germinate earlier on MG fruit. At 3 dpi, some hyphal growth was also observed for B. cinerea. Together, these observations suggest that the incompatibility of the interaction between these fungi and MG tomato fruit occurs after spore germination. To provide initial support that both F. acuminatum and R. stolonifer are capable of inducing disease responses in the host, like B. cinerea, and do not merely behave as saprotrophs , we evaluated the expression of the host gene SlWRKY33 , which is well-known to be pathogen-responsive but is not induced by abiotic stresses . To test that the induction of this gene occurred only as a result of inoculation and not wounding, we included a mock-inoculated control in our analyses. The expression patterns of SlWRKY33 measured by qRT-PCR reflected the accumulation of fungal biomass and the presence of lesions in each of the treatments . At 1 dpi, expression of SlWRKY33 was induced by inoculation with both B. cinerea and F. acuminatum but not with R. stolonifer or mock inoculation in MG fruit. In RR fruit, pathogen-induced SlWRKY33 was detected for all three pathogens at greater levels than found in MG fruit.Our observations of lesion development, fungal biomass, and activation of pathogen responses led to the hypothesis that F. acuminatum and R. stolonifer display a similar necrotrophic behavior in tomato fruit as B. cinerea. Therefore, to discover pathogenicity or virulence factors in these fungi that are important for necrotrophic infections, we performed a genomewide transcriptomic analysis of inoculated fruit at both time points as well as in vitro cultures. Due to the lack of publicly available genomic data for F. acuminatum and R. stolonifer, we assembled de novo transcriptomes for both of these pathogens from our cDNA libraries following the Trinity pipeline .