Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry Identification of Metabolites in Winterberry Fruit Putatively Associated with Natural Disease Resistance to Diaporthe ilicicola.

Winterberry holly (Ilex verticillata) is an ornamental plant popularly used in landscape design and sold as cut branches for fall and winter seasonal decoration. Latent fruit rot of winterberry is an emerging disease caused by the fungus Diaporthe ilicicola, which can result in up to 100% crop loss. Diaporthe ilicicola infects open flowers in spring, but symptom onset does not occur until the end of the growing season when the fruit is fully mature. This study was conducted to identify compounds displaying significant variation in abundance during fruit maturation and that may be putatively associated with natural disease resistance observed when the fruit is immature. Winterberry 'Sparkleberry' fruits collected at four timepoints during the 2018 and 2019 seasons were extracted in methanol and analyzed using high resolution ultra-high performance liquid chromatography-tandem mass spectrometry. The results showed a distinct separation of metabolic profiles based on fruit phenological stage. The top 100 features that were differentially expressed between immature and mature fruit were selected from both electrospray ionization (ESI) (-) and ESI (+) datasets for annotation. Eleven compounds shown to decrease throughout the season included cinnamic acids, a triterpenoid, terpene lactones, stilbene glycosides, a cyanidin glycoside, and a furopyran. Nine compounds shown to accumulate throughout the season included chlorogenic acid derivatives, hydrolysable tannins, flavonoid glycosides, and a triterpene saponin. Future research will further confirm the exact identity of the compounds of interest and determine whether they are biologically active toward D. ilicicola or I. verticillata. The results could inform breeding programs, chemical management programs, and novel antifungal compound development pipelines.

Understanding Environmental and Physiological Factors Affecting the Biology of Diaporthe ilicicola, the Fungus Causing Latent Fruit Rot in Winterberry.

Fruit rot in winterberry is associated with a complex of fungal pathogens. Among them, Diaporthe ilicicola plays a unique role by infecting flowers at bloom, resulting in symptom development in mature fruit. This research aimed to identify at what stage of maturation Ilex fruit can develop disease symptoms and correlate changes in fruit physiology (sugar and phenolic content) and environment (temperature and light intensity) with disease incidence. Correlation data informed in vitro studies testing the ability of putative factors to alter growth of D. ilicicola and select opportunistic fungi within the fruit rot complex: Alternaria alternata, Colletotrichum fioriniae, and Epicoccum nigrum. Results indicated that Ilex fruit do not develop symptoms until 81 to 108 days after inoculation. Temperature and fruit phenolic content were negatively correlated with disease incidence, while fruit sugar concentration and light intensity were positively correlated. In vitro assays revealed that sugar concentration had no effect on the growth of D. ilicicola, but increased light intensity increased hyphal growth and pycnidium formation. Additionally, phenolics extracted from fruit inhibited spore germination in A. alternata, induced secondary conidiation in C. fioriniae, and late season phenolic extracts increased hyphal melanization and pycnidial formation in D. ilicicola. Finally, drops in field temperatures, when replicated in vitro, resulted in a decrease in hyphal growth and spore germination for all fungi. These results suggest that changes in Ilex fruit phenolics during maturation and the increased exposure to light following defoliation may play a role in symptom development by altering D. ilicicola growth within the fruit.

Determining the Sources of Primary and Secondary Inoculum and Seasonal Inoculum Dynamics of Fungal Pathogens Causing Fruit Rot of Deciduous Holly.

Fruit rot of deciduous holly, caused by species of the genera Alternaria, Colletotrichum, Diaporthe, and Epicoccum, is affecting plant production in Midwestern and Eastern U.S. nurseries. To determine the sources of inoculum, dormant twigs and mummified fruit were collected, and leaf spot development was monitored throughout the season from three Ohio nurseries over two consecutive years. Mummified fruit was the main source of primary inoculum for species of Alternaria and Epicoccum, whereas mummified fruit and bark were equally important for species of Colletotrichum and Diaporthe. Brown, irregular leaf spots developed in the summer, and disease incidence and severity increased along with leaf and fruit development. Coalesced leaf spots eventually resulted in early plant defoliation. When tested for their pathogenicity on fruit, leaf spot isolates were able to infect wounded mature fruit and induce rot symptoms, which indicated that leaf spots could serve as a source of secondary inoculum for fruit infections. In addition, spore traps were used to monitor seasonal inoculum abundance in the nurseries. Fruit rot pathogens were captured by the spore traps throughout the season, with peak dissemination occurring during flowering. In this study, we also attempted to understand the role of environmental factors on leaf spot development. Although leaf spot incidence and severity were negatively correlated to mean maximum, minimum and average temperature, a decrease in temperature also coincided with leaf senescence. The role of temperature on leaf spot development should be further studied to fully interpret these results.

Investigations on the Timing of Fruit Infection by Fungal Pathogens Causing Fruit Rot of Deciduous Holly.

Fruit rot of deciduous holly is an emerging fungal disease that is affecting plant production across midwestern and eastern U.S. nurseries. To determine the growth stage(s) of host susceptibility to infection by the major pathogens associated with the disease, Alternaria alternata and Diaporthe ilicicola, and minor pathogens such as Colletotrichum fioriniae and Epicoccum nigrum, we conducted two sets of experiments over two consecutive seasons. In the first case we monitored the presence of the pathogens as well as disease progression in a commercial nursery under natural conditions by collecting plant tissues from the flower bud stage until fruit maturity. The target pathogens were consistently isolated from asymptomatic samples at all stages of fruit development and from symptomatic samples at fruit maturity across the 2 years of collection. A significant increase in fungal isolation frequency, primarily species of Alternaria and Colletotrichum, was observed right after flowering, but fruit rot symptoms only developed on mature fruit. In the second case we artificially inoculated containerized plants maintained outdoor at our research farm with individual or combined pathogens at different fruit developmental stages, and we assessed disease incidence on mature fruit to determine the time of host susceptibility to infection and, indirectly, whether pathogens in the fungal complex carry out latent infections. D. ilicicola could cause latent infection on deciduous holly fruit when inoculated at the full bloom and petal fall stages, and all inoculations made on wounded mature fruit resulted in fruit rot. These findings suggest that flowering represents a critical period to manage D. ilicicola infections and that mature fruit should be protected from any injury to avoid disease. In both experiments a negative correlation between disease incidence and temperature was found; however, the decrease in temperature also coincided with fruit ripening. The effects of temperature and changes in physiological properties of the fruit during maturation on disease development should be further investigated to fully interpret these findings.

Optimization of Storage Conditions of the Medicinal Herb Ilex asprella against the Sterigmatocystin Producer Aspergillus versicolor Using Response Surface Methodology.

The root of Ilex asprella is a commonly used herb in Southern China, and also constitutes the main raw material of Canton herbal tea. I. asprella is readily contaminated by mildew because of rich nutrients. Aspergillus versicolor producing sterigmatocystin is one of the most common molds that contaminate foodstuffs and medicinal herbs. Previous study on the evaluation of fungal contamination on samples of I. asprella revealed that A. versicolor was the dominant contaminant. In this study, experiments based on response surface methodology combined with central composite design were carried out to determine the optimal storage conditions of I. asprella to minimize the contamination of sterigmatocystin. The herb, manually innoculated with A. versicolor, was stored under different temperatures (20⁻40 °C) and humidity (80⁻95%) for seven days. The effects of temperature and humidity were evaluated using total saponin, polysaccharide and the sterigmatocystin levels as quality indexes. The results showed that A. versicolor grew quickly and produced large amounts of sterigmatocystin on I. asprella, at humidity ranging from 85% to 90% and temperatures above 26 °C. Meanwhile, total saponin and polysaccharide amounts were reduced significantly. These findings suggested that I. asprella samples should be stored in an environment with humidity and temperature below 85% and 26 °C, respectively, to reduce A. versicolor growth and sterigmatocystin production.

Identification and Characterization of Fungal Pathogens Causing Fruit Rot of Deciduous Holly.

Cut branches of deciduous holly (Ilex spp. L.) harboring colorful berries are traditionally used as ornaments in holiday decorations. Since 2012, a fruit rot of unspecified cause has resulted in significant yield reduction and economic losses across Midwestern and Eastern U.S. nurseries. In this study, symptomatic fruit samples collected from nine different locations over five years were analyzed, and several fungal species were isolated. A combination of morphological characterization, multilocus phylogenetic analyses, and pathogenicity assays revealed that Alternaria alternata and Diaporthe ilicicola sp. nov. were the primary pathogens associated with symptomatic fruit. Other fungi including A. arborescens, Colletotrichum fioriniae, C. nymphaeae, Epicoccum nigrum, and species in the D. eres species complex appeared to be minor pathogens in this disease complex. In detached fruit pathogenicity assays testing the role of wounding and inoculum concentration on disease development, disease incidence and severity increased when fruit was wounded and inoculated with a higher inoculum concentration. These findings indicate that management strategies that can protect fruit from injury or reduce inoculum may lower disease levels in the field. This research established the basis for further studies on this emerging disease and the design of research-based management strategies. To our knowledge, it also represents the first report of species of Alternaria, Colletotrichum, Diaporthe, and Epicoccum causing fruit rot of deciduous holly.

[Screening probiotic endophytic bacteria from medicinal plant flex cornuta and the phytopathogen-inhibiting effect].

Culturable endophytic bacteria were isolated from medicinal plant Ilex cornuta by plate-spreading method, strains with strong inhibitory effect on phytopathogen were screened by confrontation culture and fermentation filtrate culture methods, and the morphological changes of phytopathogen hyphae treated with endophytic bacteria were examined by microscopy and micrograph. Their phylogenetic relationships were determined by homology analysis of the 16S rDNA sequences of PCR products and the taxonomic status of the selected strains was determined based on their morphology, physiology, biochemical test results and 16S rDNA sequence analysis. A total of 85 endophytic bacteria were isolated from the healthy roots, stems, leaves and fruits of I. cornuta, and 10 strains of them showed strong inhibitory effect on Alternaria alternata, Magnaporthe grisea, Fusarium oxysporum, and were preliminarily identified belonging to four genera and seven species. Three strains with the strongest inhibitory effect, GG78 (60.3%), GG31 (48.1%) and GG13 (61.0%) belonged to Enterobacter cloacae, Enterobacter ludwigii and Bacillus cereus, respectively. Microscopic analyses showed that the inhibited phytopathogen hyphae became deformed, distorted, and partially expanded forming plasma concentration and hair-like branch on the hyphae base. These morphological changes could be caused by the extracellular metabolic substances secreted by the endophytic bacteria, such as antibiotics, hydrolytic enzymes, alkaloids and so on.

[Natural ocurrence of entomopathogenic fungi in soils cultivated with Paraguay tea (Ilex paraguariensis St. Hil.) in Misiones, Argentina]. / Ocurrencia natural de hongos entomopatógenos en suelos cultivados con yerba mate (Ilex paraguariensis St. Hil.) en Misiones, Argentina.

This study aimed to morphologically isolate, identify and characterize entomopathogenic fungi present in soils cultivated with Paraguay tea (Ilex paraguariensis). A survey of native entomopathogenic fungi was conducted from 40 soil samples grown with Paraguay tea in the province of Misiones, Argentina, from May 2008 to June 2010. The soil dilution plate methodology on selective culture media was used to isolate microorganisms. Taxonomic identification was performed using macroscopic and microscopic characters and specific keys. Twenty nine strains, belonging to the species Beauveria bassiana (n = 17), Metarhizium anisopliae (n = 2) and Purpureocillium lilacinum (n = 10) were isolated and identified.

Meroterpenes with toll-like receptor 3 regulating activity from the endophytic fungus Guignardia mangiferae.

The endophytic fungus Guignardia mangiferae isolated from Ilex cornuta leaves was shown to produce a family of meroterpenes with toll-like receptor 3 regulating activity (1-9), of which 1-3 possessed new structures. The absolute stereochemistry of 1-3 was assigned through a combination of nuclear magnetic resonance experiments, chemical derivation, CD spectra, and single-crystal X-ray diffraction analyses (CuK α ). The precursor labeled cultivation suggests that these meroterpenes are most likely assembled through terpenoid-shikimate pathways. Moreover, meroterpenes 1-3, 5-7, and 9 selectively upregulate, but 4 and 8 downregulate the toll-like receptor 3 expression in mouse dendritic cells at 10.0 µM.

Bioactive aromatic derivatives from endophytic fungus, Cytospora sp.

Two new benzyl gamma-butyrolactone analogues, (R)-5-((S)-hydroxy(phenyl)-methyl)dihydrofuran-2(3H)-one (1) and its 6-acetate (2), and a new naphthalenone derivative (8), together with eight additional known aromatic derivatives, (S)-5-((S)-hydroxy(phenyl)-methyl)dihydrofuran-2(3H)-one (3), (S)-5-benzyl-dihydrofuran-2(3H)-one (4), 5-phenyl-4-oxopentanoic acid (5), gamma-oxo-benzenepentanoic acid methyl ester (6), 3-(2,5-dihydro-4-hydroxy-5-oxo-3-phenyl-2-furyl)propionic acid (7), (3R)-5-methylmellein (9), integracins A (10) and B (11) were isolated from Cytospora sp., an endophytic fungus isolated from Ilex canariensis from Gomera. The structures of these compounds were elucidated by detailed spectroscopic analysis, comparison with reported data, and chemical interconversion. The absolute configurations of the new compounds (1, 2, 8) were established on the basis of optical rotation or CD spectra analysis. Preliminary studies showed antimicrobial activity of these compounds against the fungi Microbotryum violaceum, Botrytis cinerea and Septoria tritici, the alga Chlorella fusca, and the bacterium Bacillus megaterium.

Ring B aromatic steroids from an endophytic fungus, Colletotrichum sp.

The new (22E,24R)-3-acetoxy-19(10-->6)-abeo-ergosta-5,7,9,22-tetraen-3beta-ol (1) and the known (22E,24R)-19(10-->6)-abeo-ergosta-5,7,9,22-tetraen-3beta-ol (2), two interesting ergosteroids with rare aromatized ring B, together with seven known derivatives, namely (22E,24R)-ergosta-5,7,22-trien-3beta-ol (3), (22E,24R)-ergosta-4,7,22-trien-3-one (4), (22E,24R)-ergosta-4,6,8(14),22-tetraen-3-one (5), (22E,24R)-5alpha,8alpha-epidioxyergosta-6,22-dien-3beta-ol (6), (22E,24R)-ergosta-7,22-dien-3beta,5alpha,6beta-triol (7), (22E,24R)-6-acetoxy-ergosta-7,22-dien-3beta,5alpha,6beta-triol (8), and (22E,24R)-3,6-diacetoxy-ergosta-7,22-dien-3beta,5alpha,6beta-triol (9), were isolated from Colletotrichum sp., an endophytic fungus isolated from Ilex canariensis from Gomera. The structures of these compounds were elucidated by detailed spectroscopic analysis, comparison with reported data, and chemical interconversion. The isolation of these metabolites not only displays a beautiful array of chemical diversity, but also gives insight into the biosynthetic interconnections. Preliminary studies showed antimicrobial activity of these compounds against the fungus Microbotryum violaceum, the alga Chlorella fusca, and the bacteria Escherichia coli and Bacillus megaterium.

Known and two new species of Rhytisma (Rhytismatales, Ascomycota) from China.

Three species of Rhytisma are described based on recently collected specimens from the Anhui province, China. Rhytisma anhuiense is a new species causing a serious tar spot disease on Rhododendron simsii. Rhytisma yuexiense is a new species, which develops its stromata only on fallen leaves of Rhododendron ovatum. Rhytisma himalense on Ilex fargesii is a known species and probably widely distributed in China. Including the two new species, 11 species of Rhytisma are known from China which are presented in a key.