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Plant-associated microbiomes play important roles in plant health and productivity. However, despite fruits being directly linked to plant productivity, little is known about the microbiomes of fruits and their potential association with fruit health. Here, by integrating 16S rRNA gene, ITS high-throughput sequencing data, and microbiological culturable approaches, we reported that roots and fruits (pods) of peanut, a typical plant that bears fruits underground, recruit different bacterial and fungal communities independently of cropping conditions and that the incidence of pod disease under monocropping conditions is attributed to the depletion of Bacillus genus and enrichment of Aspergillus genus in geocarposphere. On this basis, we constructed a synthetic community (SynCom) consisting of three Bacillus strains from geocarposphere soil under rotation conditions with high culturable abundance. Comparative transcriptome, microbiome profiling, and plant phytohormone signaling analysis reveal that the SynCom exhibited more effective Aspergillus growth inhibition and pod disease control than individual strain, which was underpinned by a combination of molecular mechanisms related to fungal cell proliferation interference, mycotoxins biosynthesis impairment, and jasmonic acid-mediated plant immunity activation. Overall, our results reveal the filter effect of plant organs on the microbiome and that depletion of key protective microbial community promotes the fruit disease incidence.

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AIMS: Microbial biocontrol agents have become an effective option to mitigate the harmfulness of chemical pesticides in recent years. This study demonstrates the control efficacy of Bacillus velezensis CE 100 on the anthracnose causal agent, Colletotrichum gloeosporioides. METHODS AND RESULTS: In vitro antifungal assays revealed that the culture filtrate and volatile organic compounds of B. velezensis CE 100 strongly restricted the mycelial development of C. gloeosporioides. Moreover, a bioactive compound, butyl succinate, was isolated from the n-butanol crude extract of B. velezensis CE 100 (bce), and identified by liquid chromatography-electrospray ionization hybrid ion-trap and time-of-flight mass spectrometry (LC-ESI-QTOF-MS) and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR). Treatment with purified butyl succinate at a concentration of 300 µg mL-1 strongly controlled conidial germination of C. gloeosporioides with an inhibition rate of 98.66%, whereas butyl succinate at a concentration of 400 µg mL-1 showed weak antifungal action on the mycelial growth of C. gloeosporioides with an inhibition rate of 31.25%. Scanning electron microscopy revealed that the morphologies of butyl succinate-treated hyphae and conidia of C. gloeosporioides were severely deformed with shriveled and wrinkled surfaces. Furthermore, butyl succinate was able to control carbendazim-resistant C. gloeosporioides, demonstrating that it could be a promising agent for the suppression of other carbendazim-resistant fungal pathogens. An in vivo biocontrol assay demonstrated that the strain ce 100 broth culture and butyl succinate showed higher control efficacy on apple anthracnose than bce. CONCLUSIONS: Our findings provide insight into the antifungal potential of B. velezensis  ce 100 and its butyl succinate for efficient control of phytopathogenic fungi, such as C. gloeosporiodes, in plant disease protection. This is the first study to demonstrate the antifungal potential of bacteria-derived butyl succinate for control of C. gloeosporioides.

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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.

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Ring rot is a destructive apple disease caused by Botryosphaeria dothidea. The resistance mechanism of apple plants to B. dothidea remains unclear. Here, we show that APPLE VACUOLAR PROCESSING ENZYME 4 (MdVPE4) is involved in resistance to B. dothidea. MdVPE4 silencing reduced fruit disease resistance, whereas its overexpression improved resistance. Gene expression analysis revealed that MdVPE4 influenced the expression of fruit disease resistance-related genes, such as APPLE POLYGALACTURONASE 1 (MdPG1), APPLE POLYGALACTURONASE INHIBITOR PROTEIN 1 (MdPGIP1), APPLE ENDOCHITINASE 1 (MdCHI1), and APPLE THAUMATIN-LIKE PROTEIN 1 (MdTHA1). The expression of the four genes responding to B. dothidea infection decreased in MdVPE4-silenced fruits. Further analysis demonstrated that B. dothidea infection induced MdVPE4 expression and enzyme activation in apple fruits. Moreover, MdVPE4 activity was modulated by apple cysteine proteinase inhibitor 1 (MdCPI1), which also contributed to resistance towards B. dothidea, as revealed by gene overexpression and silencing analysis. MdCPI1 interacted with MdVPE4 and inhibited its activity. However, MdCPI1 expression was decreased by B. dothidea infection. Taken together, our findings indicate that the interaction between MdVPE4 and MdCPI1 plays an important role in modulating fruit disease resistance to B. dothidea.

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ε-poly-l-lysine (ε-PL) holds a strong antibacterial property and is widely used for food preservation. However, the application of ε-PL to enhance fruit disease resistance in postharvest longans (Dimocarpus longan Lour.) has not been explored. The objective of this study was to explore the impact of ε-PL treatment on disease occurrence and energy metabolism of longans infected with Phomopsis longanae Chi (P. longanae). It was found that, in comparison with P. longanae-inoculated longans, ε-PL could decrease the fruit disease index and adenosine monophosphate (AMP) content, increase the amounts of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and energy charge, and enhance the activities of adenosine triphosphatase (ATPase) (such as H+-, Mg2+-, and Ca2+-ATPase) in the mitochondria, protoplasm, and vacuole. The results suggest that the higher levels of ATPase activity and energy status played essential roles in disease resistance of postharvest longan fruit. Therefore, the ε-PL treatment can be used as a safe and efficient postharvest method to inhibit the disease occurrence of longan fruit during storage at room temperature.

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BACKGROUND: Syzygium samarangense (Wax apple) is an important tropical fruit tree with high economic and nutrient value and is widely planted in the tropics or subtropics of Asia. Post-harvest water-soaked brown lesions were observed on mature fruits of ornamental wax apples in Chiang Rai Province, Thailand. A fungus with morphological characters, similar to Lasiodiplodia, was consistently isolated from symptomatic fruits. Phylogenetic analyses, based on ITS, LSU, TEF1-a and tub2, revealed that our isolates were closely related to, but phylogenetically distinct from, Lasiodiplodia rubropurpurea. NEW INFORMATION: Morphological comparisons indicated that pycnidia and conidiogenous cells of our strains were significantly larger than L. rubropurpurea. Comparisons of base-pair differences in the four loci confirmed that the species from wax apple was distinct from L. rubropurpurea and a new species, L. syzygii sp. nov., is introduced to accommodate it. Pathogenicity tests confirmed the newly-introduced species as the pathogen of this post-harvest water-soaked brown lesion disease on wax apples.

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Persian walnut (Juglans regia L.) fruit with preharvest anthracnose symptoms, necrotic fruit stalks, and twigs with necrotic buds, and peaks were collected in a Hungarian orchard next to Nágocs, in September 2018. Disease incidence was approximately 15% on a Hungarian bred walnut cultivar 'Milotai 10'. Similar symptoms were found on Persian walnut in other locations (eg. Milota, Érd, Sarród, and Kocs). Acervuli were observed on necrotic lesions on fruit, and twigs with pale orange conidial masses. Conidia were hyaline, unicellular, and fusiform. Morphometric measurements of conidia showed mean length ± SD × width ± SD = 15.9 ± 1.7 × 4.5 ± 0.4 µm, length/width ratio 1:0.3 (n=100). The fungus was isolated from conidial masses on potato dextrose agar (PDA) medium amended with Chlorampenicol (25 mg/L). A total of 12 isolates were obtained as pure cultures by single-spore isolations and incubated at 23°C in dark for 10 days. The colonies were white to gray or grayish-orange on the upper side and with black spots on the reverse side. The isolates showed morphological characteristics of Colletotrichum acutatum in sensu lato (Jayawardena et al. 2016). Molecular analyses were conducted to identify the exact species. Internal transcribed spacer (ITS) region, actin (ACT), and calmodulin (CAL) partial genes were amplified by ITS1F/ITS4R, ACT512F/ACT783R and CAL1/CAL2 primers (White at al. 1990, Carbone and Kohn 1999, O'Donnell et al. 2000). The sequences of ITS region (GenBank Accession Nos: MK367398-99, MK367401-02) showed 100% identity with C. godetiae sequence. Based on ACT gene (GenBank Accession Nos: MK415991-92, MK415994-95) were 100% identity with the deposited C. godetiae type strains from walnut. The obtained sequences of CAL gene (GenBank Accession Nos: MK415998-99, MK416001-02) were same and showed 100% with other C. godetiae sequences from other host plants. The fungus was identified as Colletotrichum godetiae Neerg. Pathogenicity tests were accomplished in the field and under laboratory conditions (25°C on thermostat) on 10 green 'Milotai 10' walnut fruit, and 10 walnut twigs each. Tests were conducted on living trees, collected fruit, and two-year-old twigs by inserting mycelial agar plugs (5 mm in diameter) onto wounded pericarp tissues, which were then wrapped with wet cotton and parafilm. Wounded tissues on 5 fruit and 5 two-year-old twigs were treated with non-colonized PDA plugs as noninoculated controls. After 14 d necrotic lesions 9 to 17 mm in diameter developed on fruit on living trees. Lengths of 12 to 17 mm and width of 7 to 12 mm necrosis was measured on phloem of walnut twigs, and almost two times larger in cambium. No necrosis developed around control wounds. Koch's postulates were fulfilled with the reisolation of the pathogen from symptomatic tissues, isolates were identical morphologically and by sequence analysis of ITS region, ACT, and CAL partial genes to the original isolates. Damm et al. (2012) described two C. godetiae strains associated with walnut, one isolated in Austria and another one of unknown origin. An epidemic event of walnut anthracnose caused by Colletotrichum species mainly C. godetiae was reported in France (Da Lio et al. 2018). The pathogen was isolated from nuts, buds, insects, and stems. To our knowledge, this is the first report of anthracnose of walnut fruit caused by C. godetiae in Hungary. Anthracnose caused by C. godetiae, and previously reported C. fioriniae (Varjas et al. 2019) is becoming an increasing preharvest problem on Persian walnut in Hungary.

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BACKGROUND: Brown rots are important fungal diseases of stone and pome fruits. They are caused by several Monilinia species but M. fructicola, M. laxa and M. fructigena are the most common all over the world. Although they have been intensively studied, the availability of genomic and transcriptomic data in public databases is still scant. We sequenced, assembled and annotated the transcriptomes of the three pathogens using mRNA from germinating conidia and actively growing mycelia of two isolates of opposite mating types per each species for comparative transcriptome analyses. RESULTS: Illumina sequencing was used to generate about 70 million of paired-end reads per species, that were de novo assembled in 33,861 contigs for M. fructicola, 31,103 for M. laxa and 28,890 for M. fructigena. Approximately, 50% of the assembled contigs had significant hits when blasted against the NCBI non-redundant protein database and top-hits results were represented by Botrytis cinerea, Sclerotinia sclerotiorum and Sclerotinia borealis proteins. More than 90% of the obtained sequences were complete, the percentage of duplications was always less than 14% and fragmented and missing transcripts less than 5%. Orthologous transcripts were identified by tBLASTn analysis using the B. cinerea proteome as reference. Comparative transcriptome analyses revealed 65 transcripts over-expressed (FC ≥ 8 and FDR ≤ 0.05) or unique in M. fructicola, 30 in M. laxa and 31 in M. fructigena. Transcripts were involved in processes affecting fungal development, diversity and host-pathogen interactions, such as plant cell wall-degrading and detoxifying enzymes, zinc finger transcription factors, MFS transporters, cell surface proteins, key enzymes in biosynthesis and metabolism of antibiotics and toxins, and transposable elements. CONCLUSIONS: This is the first large-scale reconstruction and annotation of the complete transcriptomes of M. fructicola, M. laxa and M. fructigena and the first comparative transcriptome analysis among the three pathogens revealing differentially expressed genes with potential important roles in metabolic and physiological processes related to fungal morphogenesis and development, diversity and pathogenesis which need further investigations. We believe that the data obtained represent a cornerstone for research aimed at improving knowledge on the population biology, physiology and plant-pathogen interactions of these important phytopathogenic fungi.

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