Colletotrichum Fioriniae Research Articles

Blueberries infected with the fungal pathogen Colletotrichum fioriniae release odors that repel Drosophila suzukii.

Spotted-wing drosophila, Drosophila suzukii, is a serious pest of thin-skinned fruits. Alternative methods to control this pest are needed to reduce insecticide use, including new repellents. Previous research demonstrated that D. suzukii adults use odor cues to avoid blueberries infected with the fungal pathogen Colletotrichum fioriniae, which causes the disease anthracnose. To identify novel D. suzukii repellents, we investigated the volatile emission from experimentally-infected fruit, which were inoculated with C. fioriniae isolates in the laboratory, and from field-collected fruit, which were naturally infected and harvested from a field. We then tested the pathogen-induced volatiles on D. suzukii adult behavior. Volatile emission was similar between all five C. fioriniae strains, with good agreement between experimentally-infected and field-collected berries. In total, 14 volatiles were found to be more abundant in infected versus uninfected fruit headspace. In multiple-choice bioassays, nine of the 14 volatiles elicited repellency responses from adult D. suzukii. These nine volatiles were further evaluated in dual choice assays, where all nine reduced fly capture by 43-96% compared to the control. The most repellent compounds tested were the esters ethyl butanoate and ethyl (E)-but-2-enoate, which were more or equally repellent to the known D. suzukii repellents 1-octen-3-ol, geosmin, and 2-pentylfuran. Dose-response assays identified concentration-dependent effects on D. suzukii repellency and oviposition when applied individually and consistent aversion observed across doses of a 1:1 blend. We report two repellents from C. fioriniae-infected blueberries that could be useful semiochemicals for the behavioral manipulation of D. suzukii in the field. © 2023 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Spore Dispersal Patterns of Colletotrichum fioriniae in Orchards and the Timing of Apple Bitter Rot Infection Periods.

Bitter rot is a major disease of apple fruit in warm and humid regions. It is caused by various species in the Colletotrichum gloeosporioides and C. acutatum species complexes, of which C. fioriniae of the C. acutatum species complex is most common in the Mid-Atlantic region of the United States. While bitter rot management begins with good cultural practices, fungicides are generally used for consistent control. Fungicides should be applied before or during infection periods, but the timing of infection is unclear due to the hemibiotrophic lifestyle of the causal species. To determine when infection periods occur, we quantified C. fioriniae spore dispersal throughout three growing seasons and compared the temporal susceptibility of apples in two seasons of field trials. Spores were detected in rainwater from bud break to leaf drop, with the highest spore quantities in the summer and early fall correlating with optimal temperatures for C. fioriniae. Late-season-inoculated fruit had more bitter rot than early-season-inoculated fruit, but this was also positively correlated with periods of optimal temperatures and moisture for infection. In the context of previous experiments, these results suggest that infection periods are primarily determined by temperature and moisture rather than apple fruit phenology. Based on the relative numbers of spores and biotrophic and necrotrophic infections, only a tiny proportion of spores establish viable biotrophic infections, but a relatively high proportion of biotrophic infections switch to necrotrophy. We suggest bitter rot management should focus on preventing initial biotrophic infections by protecting apples during weather conditions that favor infection.

First report of Colletotrichum fioriniae infections in brown marmorated stink bugs, Halyomorpha halys

An epizootic caused by fungal pathogens occurred among Halyomorpha halys, brown marmorated stink bugs, while they were overwintering, with infections also occurring after overwintering. We report that one of the two pathogens responsible was Colletotrichum fioriniae (Marcelino & Gouli) Pennycook; a species well known as a plant pathogen and endophyte and which has only previously been reported naturally infecting elongate hemlock scales, Fiorinia externa. To prove pathogenicity, H. halys adults challenged with conidia died from infections and the fungus subsequently produced conidia externally on cadavers.

Entomopathogens infecting brown marmorated stink bugs before, during, and after overwintering.

The microsporidian, Nosema maddoxi Becnel, Solter, Hajek, Huang, Sanscrainte & Estep, infects brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), populations in North America and Asia and causes decreased fitness in infected insects. This host overwinters as adults, often in aggregations in sheltered locations, and variable levels of mortality occur over the winter. We investigated pathogen prevalence in H. halys adults before, during, and after overwintering. Population level studies resulted in detection of N. maddoxi in H. halys in 6 new US states, but no difference in levels of infection by N. maddoxi in autumn versus the following spring. Halyomorpha halys that self-aggregated for overwintering in shelters deployed in the field were maintained under simulated winter conditions (4°C) for 5 months during the 2021-2022 winter and early spring, resulting in 34.6 ± 4.8% mortality. Over the 2020-2021 and 2021-2022 winters, 13.4 ± 3.5% of surviving H. halys in shelters were infected with N. maddoxi, while N. maddoxi infections were found in 33.4 ± 10.8% of moribund and dead H. halys that accumulated in shelters. A second pathogen, Colletotrichum fioriniae Marcelino & Gouli, not previously reported from H. halys, was found among 46.7 ± 7.8% of the H. halys that died while overwintering, but levels of infection decreased after overwintering. These 2 pathogens occurred as co-infections in 11.1 ± 5.9% of the fungal-infected insects that died while overwintering. Increasing levels of N. maddoxi infection caused epizootics among H. halys reared in greenhouse cages after overwintering.

Transcriptome Analysis of Resistant and Susceptible Pecan (Carya illinoinensis) Reveals the Mechanism of Resistance to Black Spot Disease (Colletotrichum fioriniae).

Pecan, Carya illinoinensis (Wangenh.) K. Koch, is an important dried fruit and woody oil tree species grown worldwide. With continuous expansion of pecan cultivation, the frequency and scope of diseases, especially black spot disease, are increasing, damaging trees and reducing yields. In this study, the key factors in resistance to black spot disease (Colletotrichum fioriniae) were investigated between the high-resistance pecan variety "Kanza" and the low-resistance variety "Mahan". Leaf anatomy and antioxidase activities confirmed much stronger resistance to black spot disease in "Kanza" than in "Mahan". Transcriptome analysis indicated that the increased expression of genes associated with defense response, oxidation-reduction, and catalytic activity was involved in disease resistance. A connection network identified a highly expressed hub gene CiFSD2 (CIL1242S0042), which might participate in redox reactions to affect disease resistance. Overexpression of CiFSD2 in tobacco inhibited enlargement of necrotic spots and increased disease resistance. Overall, the expression of differentially expressed genes differed in pecan varieties with different levels of resistance to C. fioriniae infection. In addition, the hub genes associated with black spot resistance were identified and the functions clarified. The in-depth understanding of resistance to black spot disease provides new insights for early screening of resistant varieties and molecular-assisted breeding in pecan.

First report of Colletotrichum fioriniae causing anthracnose fruit rot on Vaccinium corymbosum in Chile.

Vaccinium corymbosum L. is the most cultivated blueberry species in Chile. Chilean fruits typically take up to 50 days to reach oversea markets; therefore, controlling post-harvest pathogens is of outmost importance to maintain international food safety and quality standards. In February 2019, the Microbial Genetic Resources Bank at INIA received fruits of V. corymbosum cv. 'Brigitta Blue' from Mariquina (-39.567869, -72.992461), located in the southern Chilean blueberry production zone, for post-harvest disease diagnosis. Asymptomatic fruits were incubated in moist-chambers at 25 °C with light/darkness cycles of 12 h. After 5 d, some fruits showed sunken areas and small surface wounds that exudated orange masses of conidia; under the epidermis, gray acervuli were also detected. After 15d, these fruits became dehydrated, mummified, and covered by mycelia, all characteristic symptoms of anthracnose (Wharton and Schilder 2008). In Chile, Colletotrichum gloeosporioides has, thus far, been the only causal agent of anthracnose reported in blueberry (Lara et al. 2003). Conidia exudated from the diseased fruit were inoculated on potato-dextrose agar (PDA) and incubated at 25 °C for 7 d. The resulting colony was predominantly cottony with gray aerial mycelium, displaying masses of pale orange conidia; on the reverse side, the colony was a pink-reddish color. Under a microscope, conidia were hyaline, fusiform to elliptic in shape, and displaying guttulate of 12.2±1.2 × 4.17±0.3 μm (n=30), characteristics coinciding with those described for Colletotrichum fioriniae (Pennycook 2017; Shivas and Tan 2009) (Supplementary Figure 1). The isolate was deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM) as RGM 3330. Genomic DNA extraction of RGM 3330 and phylogenetic analyses were carried out according to Cisterna-Oyarce et al. (2022). A multi-locus sequencing analysis was carried out using five genetic markers. The internal transcribed spacer (ITS), glyceraldehyde 3-phosphate dehydrogenase (gapdh), actin (act), and chitin synthase 1 (chs-1) were PCR-amplified following Damm et al. (2012) and beta-tubulin (tub) following Glass and Donaldson (1995). Sequences were deposited in GenBank (ON364141 for ITS and ON369167-70 for tub, act, chs-1, and gapdh, respectively) (Sayers et al. 2021). A BLAST analysis carried out in SequenceServer (Priyam et al. 2019), using a custom database of sequences retrieved from Damm et al. (2012) and Liu et al. (2020), showed that all genetic markers were 100% identical to those of C. fioriniae CBS 128517T (ITS (540/540 identities), gapdh (249/249), act (245/245), and chs-1 (274/274)), except for tub, which shared 99.8% of its identities (416/417) with this species. Maximum likelihood phylogenetic estimation clustered RGM 3330 with C. fioriniae strains CBS 128517T and CBS 126526 with 100% bootstrap support (Supplementary Figure 1). Koch’s postulates were carried out with asymptomatic fruits of V. corymbosum cv. 'Brigitta Blue'. Prior to inoculation, fruits were surface-sterilized for 10 s in 70% ethanol, 3 s in 1% NaOCl, 10 s in 70% ethanol, rinsed three times with sterile distilled water, and subsequently placed in moist-chambers. Two groups of three repetitions of 20 fruits each were sprayed with 9 × 106 conidia/mL of RGM 3330 for the first group and with sterile distilled water for the control. After 5 d at 25 °C with light/darkness cycles of 12 h, only fruits sprayed with the conidial solution developed symptoms of anthracnose and the re-isolated fungi were identical in morphology to RGM 3330. This is the first report of C. fioriniae in blueberry in Chile.

Brown leaf spot of Prunus serrulata Caused by Colletotrichum fioriniae in Sichuan, China.

Prunus serrulate Lindl is widely cultivated in urban areas of China. It is mainly used for wood cultivation and urban landscaping. In May 2021, new leaf spot disease was observed in Chengdu city (30°42' to 30°45'N, 103°51' to 104°7'E), with 69.3% disease incidence, which could inhibit leaf growth and reduce their biomass. A planting area of more than 1000 square meters was investigated. The diseased leaves were mostly concentrated in the lower position of plants, where the humidity was higher. The disease infected P. serrulata leaves and occurred in the field from March to October, with the highest incidence in early May. The typical symptoms initially appeared as brown necrotic lesions on the margin of the leaves. The lesions then enlarged gradually and developed into reddish brown spots, eventually coalescing into large irregular, necrotic lesions with dark brown margins. Finally, the diseased leaves withered and died. Conidiomata were not formed on the diseased tissue. Ten symptomatic leaves were collected from 5 different trees in the planting area. Infected tissues from ten samples were cut into small pieces of 3 × 3 mm. The infected tissues were surface-sterilized by 3% sodium hypochlorite and 75% ethanol respectively for 30s and 60s, and rinsed three times in sterile water. Then they were blot-dried with autoclaved paper towels and cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/mL), and incubated at 25°C for 4 to 8 days. After culturing for 8 days at 25℃ and 12 h/12 h light/dark on PDA, the colony diameter reached 67.5 to 78.6 mm. The colonies were initially white, cottony, then became light pink to misty rose at the center, and the reverse side of the colony turned dark red to red and had pale yellowish borders. The conidia were straight, smooth-walled, colorless, fusiform with acute ends, measuring 8.2 to 16.7 × 3.1 to 5.9 μm in size (n = 100). For molecular identification, the genomic DNA of the representative isolate RBWY202105 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The internal transcribed spacer (ITS) [ITS1/ITS4 (White et al., 1990)], histone3 (HIS3) [CYLH3F/CYLH3R (Crous et al. 2004)], chitin synthase (CHS-1) [CHS-79F/CHS-345R (Carbone & Kohn, 1999)], actin (ACT) [ACT512F/ACT (Carbone & Kohn, 1999)], β-tubulin (TUB2) [BT2A/BT2B (O'Donnell et al., 1997)], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [GDF/GDR (Templeton et al. 1992)] were amplified. Sequences were deposited in GenBank (ITS: ON000436, HIS3: ON014581, CHS-1: ON014579, ACT: ON014583, TUB2: ON014582, and GAPDH: ON014580). BLAST results indicated that the ITS, HIS3, CHS-1, ACT, TUB2 and GAPDH sequences showed >99% identity with Colletotrichum fioriniae (Marcelino & Gouli) R.G. Shivas & Y.P sequences at NCBI (GenBank MW497230 (561/582), MT740312 (415/415), KU736865 (258/258), MK680659 (246/246), MK967342 (757/757), and MW656269 (263/263)). The conidial suspension (1 × 106 conidia/ml) was used for inoculation by spraying leaves of ten 4-year-old P. serrulata plants for pathogenicity test. Fifteen leaves of each plant were inoculated with spore suspensions on the leaves (600 μl per leaf). The same amount of control leaves was sprayed with sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 25°C under a 16 h/8 h photoperiod and 67 to 78% relative humidity. After ten days, the symptoms observed on the inoculated plants were similar to those of the original diseased plants, but the controls remained asymptomatic. Colletotrichum fioriniae was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis (The ITS, HIS3, TUB2, CHS-1, GAPDH and ACT genes). The pathogenicity test was repeated thrice, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of brown leaf spot on P. serrulata caused by C. fioriniae in China. The identification of C. fioriniae could provide relevant information for taking management strategies and further research on the Prunus serrulata disease.

First report of leaf anthracnose on the Whorled Sunflower, Helianthus verticillatus, caused by Colletotrichum fioriniae in the United States.

Whorled sunflower, Helianthus verticillatus Small, is an endangered (U.S. Fish and Wildlife Service 2014) perennial sunflower species indigenous to the southern United States (Matthews et al. 2002; Ellis et al. 2008). Helianthus verticillatus has a showy yellow floral display in the Fall that attracts a diversity of insect visitors (Strange et al. 2020). Its hardiness in the landscape and late-season blooming makes it a potential ornamental (Trigiano et al. 2021). In June 2021, anthracnose-like lesions were observed on mature leaves collected from potted H. verticillatus plants grown in the nursery compound at the University of Tennessee, Knoxville, TN, USA. Irregularly shaped leaf spots with 1‒2 mm tan centers were observed on mature leaves, which later expanded to 3‒5 mm, and became dark brown- to- black surrounded by chlorotic halos (Fig.1). Lesions from three infected leaves were excised from a single potted plant, trimmed to 1.5-cm squares with green borders, and surface-sterilized (Trigiano et al. 2018). Tissues were placed onto potato dextrose agar (PDA), amended with 100 mg/ml of each streptomycin sulfate and chlorotetracycline, and incubated at 21 °C in the dark until axenic cultures were obtained. Initially, appressed white- to- pale gray mycelia were formed that turned light pinkish-orange with age (Fig. 2A). Conidia (Fig. 2B-C) were single-celled, hyaline, and cylindrical- to- fusiform with acute ends, and were similar to Colletotrichum fioriniae (Damm et al. 2012). Conidia measured 8.9 ± 1.3 μm long and 3.3 ± 0.6 μm wide (N=40). Genomic DNA was isolated with a Phire Direct Plant PCR kit (Thermo FisherScientific, Waltham, MA). The partial beta-tubulin (TUB2) gene, chitin synthase 1 (CHS-1) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and the internal transcribed spacer (ITS) region of ribosomal DNA were amplified with primers T1/BT2B, CHS-354R/CHS-79F, GDF1/GDR1, and ITS1/ITS4, respectively and sequenced (Damm et al. 2012). The resulting sequences were submitted to GenBank (TUB2, ON036471; CHS-1, ON036472; GAPDH, ON036470; and ITS, ON008206). Consensus sequences had 100% identity with C. fioriniae type culture CBS 128517 accessions JQ949943 (TUB2), JQ948953 (CHS-1), JQ948622 (GAPDH), and MH865005 (ITS rDNA). Because H. verticillatus is endangered, and the scarcity of available plant material, Koch's postulates were performed using a detached leaf assay (Boggess et al. 2022). Six healthy leaves were surface-sterilized using the previously described protocol, longitudinally bisected, and placed on 1.5% water agar in three 15 × 100 mm petri dishes. Three half leaves were inoculated with sterile, 5 mm-diameter PDA plugs (controls). The remaining three leaves were inoculated with 5 mm-diameter PDA plugs of C. fioriniae and incubated as described previously. After ten days, necrotic lesions developed on leaves inoculated with C. fioriniae and were similar to the initially observed lesions on plants. Lesions did not develop on control leaves. Colletotrichum fioriniae was re-isolated from lesions using the previously described protocol. The disease does not appear to cause mortality of H. verticillatus and does not require control measures but does reduce the aesthetic value of the plant. To the best of our knowledge, this is the first report of C. fioriniae infecting H. verticillatus in the United States.

First report of Colletotrichum fioriniae causing anthracnose on pear fruit in Serbia.

European pear (Pyrus communis L.) is one of the most important fruits in Serbia, with a total production of 55,938 tonnes in 2021 (Statistical Office of the Republic of Serbia). In November 2021, pear fruits (cv. Santa Maria) with typical anthracnose symptoms were collected in one storage facility in the area of Smederevo, Serbia. The disease incidence was about 2%. A total of nine infected fruits had dark, circular, sunken necrotic lesions with concentric rings of orange conidial masses produced in black acervuli. The tissue pieces from the edges of lesions (3 to 5 mm2) were surface-sterilized in 1% sodium hypochlorite for 1 min, rinsed twice in sterilized distilled water, air-dried, and incubated on potato dextrose agar (PDA) at 25°C. One isolate/diseased fruit was obtained (nine in total). Colony morphology of all isolates on PDA was light gray to pinkish on the upper side, and pink to vinaceous on reverse after 7 days of incubation at 25°C in the dark. Average growth rate was 64 ± 2 mm. The conidia were hyaline, fusiform, aseptate, smooth-walled, with both ends acute, and measured 12.8 to 16.6 µm × 3.5 to 4.5 µm (n =100). Appressoria were single, smooth-walled, dark brown, clavate to irregular outline, 5.5 to 9.6 × 4.8 to 7.2 μm (n =20). These morphological characterstics are consistent with the description of Damm et al. (2012) for Colletotrichum fioriniae. To confirm this identification, genomic DNA of representative isolate SZ-21-36 was extracted and the ITS region of the rDNA, partial beta-tubulin gene (BenA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and partial actin gene (ACT) were amplified using the primers ITS1/ITS4, Bt2a/Bt2b, GDF1/GDR1, and ACT512F/ACT783R, respectively (Hassan et al. 2019). The nucleotide sequence of each locus has been deposited in GenBank under Accession Nos. ON171625 (ITS), ON186696 (BenA), ON186697 (GAPDH), and ON186698 (ACT). Multilocus phylogenetic analysis based on the above mentioned molecular markers placed Serbian isolate of C. fioriniae with other isolates of this species deposited in GenBank (bootstrap support of 100%). Pathogenicity test was conducted on symptomless, detached pear fruits (cv. Santa Maria). Five surface-sterilized fruits were wound-inoculated with 50 μl of a conidial suspension (1 × 106 conidia/ml). After 10 days of incubation in a plastic chamber (25°C, 90% relative humidity, and 12-h photoperiod), typical anthracnose lesions developed on inoculated fruits. Five control fruits inoculated with sterile distilled water remained asymptomatic. The experiment was repeated once. Fungal colonies reisolated from the lesions showed the same morphological features as the original isolate. Pathogen was not reisolated from the control fruits. C. fioriniae was previously reported on European pear fruits in Croatia (Ivić et al. 2013), France (Da Lio et al. 2017), China (Fu et el. 2019), and in the USA (Pavlović et al. 2019). To our knowledge, this is the first report of anthracnose of pear fruit caused by C. fioriniae in Serbia. Currently, the economic impact of anthracnose caused by this pathogen is minimal, but its presence decreases commercial value and quality of pear fruits, and can be a risk to the further spread of C. fioriniae to other plant species.

Thymol Edible Coating Controls Postharvest Anthracnose by Regulating the Synthesis Pathway of Okra Lignin.

Okra has received extensive attention due to its high nutritional value and remarkable functional characteristics, but postharvest diseases have severely limited its application. It is important to further explore the methods and potential methods to control the postharvest diseases of okra. In this study, Colletotrichum fioriniae is the major pathogen that causes okra anthracnose, which can be isolated from naturally decaying okra. The pathogenicity of C. fioriniae against okra was preliminarily verified, and the related biological characteristics were explored. At the same time, an observational study was conducted to investigate the in vitro antifungal effect of thymol edible coating (TKL) on C. fioriniae. After culturing at 28 °C for 5 days, it was found that TKL showed an obvious growth inhibition effect on C. fioriniae. The concentration for 50% of the maximal effect was 95.10 mg/L, and the minimum inhibitory concentration was 1000 mg/L. In addition, it was found that thymol edible coating with a thymol concentration of 100 mg/L (TKL100) may cause different degrees of damage to the cell membrane, cell wall, and metabolism of C. fioriniae, thereby inhibiting the growth of hyphae and causing hyphal rupture. Refer to the results of the in vitro bacteriostatic experiment. Furthermore, the okra was sprayed with TKL100. It was found that the TKL100 coating could significantly inhibit the infection of C. fioriniae to okra, reduce the rate of brown spots and fold on the okra surface, and inhibit mycelium growth. In addition, the contents of total phenols and flavonoids of okra treated with TKL100 were higher than those of the control group. Meanwhile, the activities of phenylalaninammo-nialyase, cinnamic acid-4-hydroxylase, and 4-coumarate-CoA ligase in the lignin synthesis pathway were generally increased, especially after 6 days in a 28 °C incubator. The lignin content of TKL-W was the highest, reaching 65.62 ± 0.68 mg/g, which was 2.24 times of that of CK-W. Therefore, TKL may promote the synthesis of total phenols and flavonoids in okra, then stimulate the activity of key enzymes in the lignin synthesis pathway, and finally regulate the synthesis of lignin in okra. Thus, TKL could have a certain controlling effect on okra anthracnose.

Fungal pathogens causing postharvest fruit rot of wolfberry and inhibitory effect of 2,3-butanedione.

Fungal pathogen contamination is one of the most important factors affecting the postharvest quality and shelf life of wolfberry fruits. Therefore, the prevention and control of fungal pathogens that cause fruit rot has become particularly important. Volatile antifungal agents of biological origin have broad application prospects. They may be safer and more efficient than traditional physical and chemical methods. Four pathogenic fungi were isolated and purified from rotting wolfberry. These pathogenic fungi were determined to be Mucor circinelloides LB1, Fusarium arcuatisporum LB5, Alternaria iridiaustralis LB7, and Colletotrichum fioriniae LB8. In vitro fumigation experiments showed that 2,3-butanedione can effectively inhibit the mycelial growth, spore germination, and sporulation ability of pathogenic fungi. The scanning electron microscope (SEM) showed morphological changes in hyphae. Propidium iodide (PI) Staining and leakage of 260 and 280 nm-absorbing increased, suggesting damage to cell membranes. Furthermore, 2,3-butanedione was found to significantly improve fruit firmness, soluble solid, total phenol, flavonoid, and soluble sugar content, as well as higher SOD enzyme activity and lower PPO and POD enzyme activity in the treated fruit, indicating that 2,3-butanedione can effectively reduce the adverse effects of pathogenic fungi in wolfberry. Based on these results, we conclude that 2,3-butanedione is effective against infection by pathogenic fungi in post-harvest wolfberry. 2,3-butanedione should be considered a viable substitute for conventional fungicides that are currently used to control rot in wolfberry.

First Report of Colletotrichum fioriniae Causing Grapevine Anthracnose in New York.

Grapevine is one of the most widely-planted fruit crops in the world, and is the most economically important fruit crop in the state of New York, USA. Symptoms of anthracnose on grapevine are similarly widely-reported on grapevine fruit and foliage, and such symptoms are commonly attributed to Elsinöe ampelina (Wilcox et al., 2015). However, similar symptoms, if not identical, to those associated with E. ampelina have been sporadically attributed to various species in the genus Colletotrichum. In September 2021, a survey was conducted in three research vineyards at Cornell AgriTech in Geneva, NY. Symptoms of anthracnose werebserved on four Vitis interspecific hybrid breeding lines in a 1 ha vineyard. Leaves, fruit, and petioles showing symptoms of anthracnose, i.e., sunken necrotic lesions with grayish centers and brownish margins, were collected. Symptomatic and healthy portions of surface-sterilized tissues were placed on PDA medium and incubated at 23oC for 7 days. Several petiole samples yielded colonies of white to greyish mycelium, with some red to orange pigmentation (Fig. 1A and 1B), similar to those described by Chowdappa et al. (2009) for Colletotrichum species isolated from grapevine in India. Cultures were allowed to sporulate. Slides from cultures were prepared and examined at 400X magnification. Conidia from cultures were cylindrical with rounded ends, 13.5-15.2 μm in length and 7.6-9.0 μm in width (Fig. 1C). Koch's postulates were fulfilled by inoculating detached healthy leaves of V. vinifera 'Chardonnay' that had been surface sterilized in 10% sodium hypochlorite and triple-rinsed in sterile distilled water. Drop inoculation was used from a suspension of 105 conidia/ml from the foregoing pure cultures as five 2 µL droplets per leaf. Inoculated detached leaves were maintained on water agar in a Petri dish at 23oC. Four days after inoculation, symptoms were observed and compared with the originally collected samples. Inoculated leaves displayed symptoms typically found on the collected tissues, and the original pathogen, as confirmed by colony morphology and conidial characteristics and dimensions, was reisolated from inoculated leaves, and not from non-inoculated controls. For molecular characterization, fungal DNA was isolated by using Qiagen DNeasy kit and amplified using the following primer pairs: ITS1/ITS4, TEF (Hyun et al., 2009), E. ampelina F/R (Santos et al. 2018), TUB2, ACT, HIS3, GAPDH and CHS1 (Damm et al., 2001). PCR products were purified using ExoSAP-IT, and samples were Sanger sequenced. Sequences were analyzed using Geneious Prime software, and the resulting sequences (NCBI accessions OL720215, OL720216, OL720217, OL720218, OL853836, OM982612, OM982613, OM982614, OM982615 and OM982616) had 94 to 100% identity to Colletotrichum fioriniae NCBI accessions MN944922.1, MK646015.1, MN944922.1, MN856415.1, KU847413.1, MN520490.1, MN544294.1, KY695259.1, MN535117.1 and MN544295.1. Symptoms of grapevine anthracnose caused by Colletotrichum species have been reported from India (Chowdappa et al., 2009) and Korea (Kim et al., 2021). To our knowledge this is the first report of grapevine anthracnose caused by C. fioriniae Anthracnose and ripe rot are diseases of increasing importance, particularly as new grapevine cultivars with resistance to powdery mildew or downy mildew are adopted. Taxonomy of the causal agents (E. ampelina and Colletotrichum spp.) has undergone considerable revision. Consequently, distribution and relative prevalence of the various taxa will require further study.

Identification and characterization of Colletotrichum fioriniae and C. fructicola that cause anthracnose in pecan.

Pecan (Carya illinoinensis Wang. K. Koch) is a deciduous tree of the Juglandaceae family with important economic value worldwide. Anthracnose of the pecan leaves and shuck is a devastating disease faced by pecan-growing areas in China. However, the causal species occurring on pecan remain largely unidentified. we collected samples of diseased pecan from the provinces of China, Leaves and fruits affected by anthracnose were sampled and subjected to fungus isolation, The morphological characters of all strains were observed and compared; Multi-locus phylogenetic analyses [Internally transcribed spacer (ITS), Actin (ACT), Calmodulin (CAL), Chitin synthase (CHS1), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and b-tubulin (TUB2)] were performed on selected representative strains; examine their pathogenicity on leaves of pecan.The results showed that: (1) resulting in a total of 11 Colletotrichum isolates, Two Colletotrichum species were identifified to be C. fioriniae and C. fructicola; (2) Pathogenicity tests revealed that both species caused black spots on pecan leaves and fruit, The virulence of the different isolates varied substantially, with C. fioriniae PCJD179 being the most virulent; (3) The susceptibility levels of pecan tree varieties, 'Mahan' and 'Kanza', were determined, No significant differences were observed in the lesion sizes produced by the various isolates in 'Kanza', while there were signifificant differences in 'Mahan'. This study is thefifirst to determine that C. fructicola and C. fioriniaecan cause anthracnose in pecan in China. It improves the understanding of the species that cause anthracnose in pecan and provides useful information for the effective control of this disease in China.

First report of Colletotrichum fioriniae associated with olive anthracnose in Italy

First report of Colletotrichum fioriniae associated with olive anthracnose in Italy

Functional characterization of MANNOSE-BINDING LECTIN 1, a G-type lectin gene family member, in response to fungal pathogens of strawberry.

The mannose-binding lectin gene MANNOSE-BINDING LECTIN 1 (MBL1) is a member of the G-type lectin family and is involved in defense in strawberry (Fragaria × ananassa). Genome-wide identification of the G-type lectin family was carried out in woodland strawberry, F. vesca, and 133 G-lectin genes were found. Their expression profiles were retrieved from available databases and indicated that many are actively expressed during plant development or interaction with pathogens. We selected MBL1 for further investigation and generated stable transgenic FaMBL1-overexpressing plants of F. ×ananassa to examine the role of this gene in defense. Plants were selected and evaluated for their contents of disease-related phytohormones and their reaction to biotic stresses, and this revealed that jasmonic acid decreased in the overexpressing lines compared with the wild-type (WT). Petioles of the overexpressing lines inoculated with Colletotrichum fioriniae had lower disease incidence than the WT, and leaves of these lines challenged by Botrytis cinerea showed significantly smaller lesion diameters than the WT and higher expression of CLASS II CHITINASE 2-1. Our results indicate that FaMBL1 plays important roles in strawberry response to fungal diseases caused by C. fioriniae and B. cinerea.

First Report of Colletotrichum fioriniae Causing Anthracnose on Rhododendron delavayi in China.

Rhododendron delavayi Franch, a member of Ericaceae family, is globally famous for its garden flowers with significant ornamental value (Liu et al., 2020). In July 2020 and 2021, a disease survey of R. delavayi groves was conducted in Baili Azalea Forest Area (N27°10'-27°20', E 105°04'-106°04'). We arbitrarily selected an area with around 280 R. delavayi trees covering 2.5 hectares in R. delavayi grove where 20-35% of leaves showed symptoms of anthracnose. Typical symptoms included elliptical to irregularly shaped brown lesions on leaves and masses of black dots clustered on it. About 30 pieces of leaves with anthracnose lesions were collected. A few black dots were picked from the lesions with a sterilized needle, plated on water agar and incubated at 25℃ for 12 h to observe spore germination (Fang, 2007). Then the germinated spores were transferred onto PDA medium for further purification and morphological observation. Fourteen single-spore isolates with similar morphology were obtained. The surface of the colony was white or gray and spongy; the edge was smooth; and the back side was pinkish brown after 7 days of growth on PDA. Conidia were spindle-shaped, transparent, 11.1-16.6×3.6-4.9 μm (n=50). Appressorium from conidia was nearly ovate or proximate, brown or dark brown in color, 4.3-10.3 ×3.2-7.6 μm (n=50). These characteristics are consistent with Colletotrichum fioriniae reported by Shivas and Tan (2009). DNA was extracted from a representative isolate MYDJ12. The internal transcribed spacer region (ITS), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB), actin (ACT), and chitin synthase 1 (CHS-1) genes were amplified using primer pairs described by Damm et al. (2012). The sequences were deposited in GenBank with accession number MW692854 (ITS), MW727518 (GAPDH), MW727519 (TUB2), MW727520 (ACT), and MW727521 (CHS-1). BLASTN searches of the ITS, GADPH, TUB2, ACT and CHS-1 genes revealed 100% (540/540 nucleotides), 100% (254/254 nucleotides), 99.38% (4488/491 nucleotides), 98.77% (242/245 nucleotides) and 100% (282/282 nucleotides) homology with those of C. fioriniae CBS:128517T in GenBank (NR_111747, JQ948622, JQ949943, JQ949613 and JQ948953 respectively). The phylogenetic tree showed the isolate MYDJ12 to cluster with C. fioriniae CBS:128517T. Finally, two-year old R. delavayi plants (n=5) were inoculated by wounding with a syringe needle and placing 10 μL of spore suspension (106 spores per mL) of the isolate MYDJ12 on three leaves per plant. Control leaves were inoculated with sterile water. The experiment was conducted twice. Inoculated leaves were wrapped in parafilm tape and then the plants were placed in a greenhouse at 25°C with high relative humidity (90 to 95%). Seven days after incubation, brown lesions appeared, similar to those observed in the grove. Black dots clustered on the lesions after 15 days. Re-isolation was conducted 20 days after inoculation. From all the five inoculated plants, similar symptoms were observed, and the same pathogen was re-isolated. One of the isolates was selected for morphological observation and multi-gene (ITS, GAPDH, ACT, TUB2 and CHS-1) analysis indicated the reisolated fungus to be C. fioriniae. No fungal pathogens were isolated from mock inoculated plants. This study can provide effective management and useful information for the control of this disease on R. delavayi in Baili Azalea Forest Area.

First report of Colletotrichum fioriniae causing leaf spot on Schima superba in the world.

Schima superba Gardn. et Champ. is a subtropical evergreen tree species naturally distributed mainly in China, Japan, and Vietnam. It is primarily planted for its timber and urban landscaping in China (Ni, 1996). In September 2018, leaves necrotic spots were observed on S. superba in Jiangxi Forest Breeding Center (28°57'19.52" N, 115°39'21.32" E), Jiangxi Province, China. The disease incidence was about 30%. Initially, spots were circular to semicircular, grayish-brown in the center with dark brown margin, then expanded and eventually collapsed into sunken necrotic lesions. To identify the agent, diseased leaves were collected randomly. Pieces (5 × 5 mm) from the lesion borders were surfaced sterilized in 70% ethanol (30 s), 3% NaOCl (60 s), and rinsed 3 times in sterile water. These pieces were put on potato dextrose agar (PDA) and cultured at 25 °C. Pure cultures were obtained by monosporic isolation, and 3 isolates (MH-1, MH-2, MH-3) were used for morphological studies and phylogenetic analyses. On PDA, colonies were initially white, cottony, then became pinkish to deep-pink at the center and pink on the reverse. Conidia were fusiform with acute ends, smooth-walled, hyaline, 13.7-18.5 × 4.6-6.1 µm (16.4 ± 1.3× 5.3 ± 0.6 µm, n = 100). Conidiophores were colorless to pale brown, smooth, septate. Conidiogenous cells were colorless to pale brown, smooth, cylindrical to ampulliform. The morphological characteristics fit the descriptions of Colletotrichum acutatum J. H. Simmonds sensu lato (Damm et al., 2012). For accurate identification, genomic DNA of 3 isolates was extracted, and the internal transcribed spacer (ITS), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin 2 (TUB2), and chitin synthase (CHS-1) were amplified and sequenced using the corresponding primers (Weir et al., 2012). The sequences were deposited in GenBank (ITS: MZ325946, MZ325947, MW584318; ACT: MZ399375, MZ419566, MW661171; CHS-1: MZ399376, MZ419567, MW661172; MZ399377, GAPDH: MZ419568, MW661173; TUB2: MZ399378, MZ419569, MW661174). Five loci were concatenated, and the aligned sequences (1528bp) were 99.89% homologous to ex-type C. fioriniae (Marcelino & Gouli) R. G. Shivas & Y. P. Tan CBS128517. Phylogenetic analysis using the maximum likelihood showed that 3 isolates were clustered in C. fioriniae clade with 100% bootstrap support. Based on the multi-locus phylogeny and morphology, 3 isolates were identified as C. fioriniae. Pathogenicity tests were performed on 36 seedlings of S. superba (2-year-old). The leaves were wounded slightly and inoculated with a drop of spore suspension (106 conidia/mL). The sterile water was used as controls. All the tested leaves were covered with black plastic bags to keep them moist for 2 days. All seedlings were placed in the greenhouse (25 °C, 12 h light/dark) for 10 days, and all inoculated leaves had typical symptoms. The controls were asymptomatic. The same fungus was reisolated from the lesions, fulfilling Koch's postulates. Colletotrichum fioriniae was described as a new species from the C. acutatum s. l. (Shivas et al., 2009), and it was an important plant pathogen, such as Pyrus spp. (Pavlović et al., 2019), Morus alba L. (Xue et al., 2019), and so on. This is the first report of the newly emerging disease of S. superba caused by C. fioriniae in the world, and its potential threat should be evaluated in the future. This study provided crucial information for epidemiologic studies and appropriate control strategies.

Infestation of red currant berries and its varieties by fungi of genus Colletotrichum Sacc. in Russia

Some species of parasitic fungi from the genus Colletotrichum afflict the vegetative organs of a plant, while the others affect the reproductive organs. This results in the deterioration of berry quality and yield and the potential death of a plant. Colletotrichum fungi causing anthracnose in the red currant berry were first isolated in Russia from the fruits of red currant (Ribes rubrum) and its varieties received from the Moscow, Yaroslavl and Kostroma regions and the Mari El Republic. The susceptibility of these crop varieties depended on the infection potential of Colletotrichums pp. in the plantings and weather conditions. Infestation symptoms include wrinkled fruit that develops during berry ripening. Species of this pathogen are preserved within mummy berries and dry clusters on the branches. The flowers were infected under the conditions of the Moscow Region in the first decade of May 2021; under the conditions of continental climate in the Mari El Republic, visual symptoms of the disease were absent within this period; however, the pathogen had already been present in the plantations. Therefore, it can be concluded that the pathogens behave as biotrophs when infecting red currant. The results of morphological and molecular genetic analyses showed that two species of the genus Colletotrichum parasitise on red currant in the Moscow region: Colletotrichum godetiae Neerg., Friesia and Colletotrichum fioriniae Marcelino&Gouli.

First Report of Cinnamomum japonicum Anthracnose Caused by Colletotrichum fioriniae in Sichuan, China.

The Cinnamomum japonicum Sieb is widely cultivated in urban in China. It's used to make essential, lubricant, soap, and waterproof timber. In September 2019, This new leaf spot was discovered in Chengdu city (30°05'to 31°26'N, 102°54'to 104°53'E), with approximately 61.20% disease incidence. The symptoms started to occur from May to June, the worst from August to September. Firstly, the typical symptom showed round or oval, brown, and slightly sunken necrotic lesions. Gradually, the necrotic lesions increased in number, and expanded; under humid conditions the central part of the spots became black and ruptured, with orange conidial masses emerged at the margin of lesions. Finally, the leaves turn yellow and fall off. Infected tissues from ten samples were cut into small pieces 2 × 2 mm, surface sterilized for 30 s in 3% sodium hypochlorite, 60 s in 75% ethanol, rinsed three times in sterile water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/mL), and incubated at 25°C in a dark. Finally, 8 typical isolates exhibited the morphology described as C. fioriniae (Amelie Grammen et al. 2019). After 5 days, the colony diameter reached 28.6 to 41.2 mm and had white to light grey aerial mycelium, but was pink at the base. Orange conidia masses formed after 6-7 days, conidia were oval, slender and fusiform with acute ends (Figure 1e, f), measuring 8.3 to 19.6 × 2.9 to 7.1 μm (average: 13.9 × 4.8 μm) (Tashiro et al. 2018; Chechi et al. 2019). For molecular identification, DNA was extracted from 8 fungal colonies using a plant genomic DNA extraction kit (Solarbio, Beijing). The 5.8S nuclear ribosomal genes with the two flanking internal transcribed spacer (ITS), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and partial sequences of the actin (ACT), chitin synthase 1 (CHS-1), beta-tubulin (TUB2), histone3 (HIS3), calmodulin (CAL) and glutamine synthetase (GS) genes were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al. 1990), GDF/GDR (Templeton et al. 1992), ACT-512F/ACT-783R, CAL 228F/CAL 737R (Carbone et al. 1999), CHS-354R/CHS-79F (Weir et al. 2012), TUB1F/Bt2bR, CYLH3F/CYLH3R (Crous et al. 2004), and GSF1/GSR1 (Liu et al. 2015), respectively. Sequences were deposited in GenBank (ITS: MT466533, GAPDH: MT460415, ACT: MT460414, CAL: MT954332, CHS-1: MT954330, TUB2: MT460416, HIS3: MT954331, and GS: MT460417). BLAST analysis showed >99.4% identity with several reference sequences of Colletotrichum fioriniae strain CBS 128517 and strain EHS58 (teleomorph of Glomerella fioriniae) previously deposited in GenBank. The conidial suspension (1 × 107 conidia/mL) collected from PDA cultures with 0.05% Tween 80 buffer was used for inoculation by spraying leaves of 5-year-old C. japonicum plants for pathogenicity test. Ten leaves of each plant (10 pots in total) were inoculated with spore suspensions (Approximately 500 μL per leaf). An equal number of control leaves were sprayed with 0.05% Tween 80 buffer to serve as a control. Twenty days later, the inoculated plants showed the similar symptoms as the original diseased plants but the controls remained asymptomatic. The C. fioriniae was re-isolated from the infected leaves and identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. To our knowledge, this is the first report of C. japonicum anthracnose caused by C. fioriniae in China.

Fungi as potential factors limiting natural regeneration of pedunculate oak (Quercus robur) in mixed‐species forest stands in Poland

AbstractDieback of youngQuercus roburseedlings can limit natural regeneration in mixed‐species forest stands in Poland. The aim of this study was to examine the role of fungi in the dieback of oak seedlings in central Europe. Fungi were isolated from the stems and roots ofQ.robur, from both healthy seedlings and seedlings with symptoms, that were sampled from four stands in Poland. In total, 111 distinct taxa were identified. Ascomycota was dominant, representing 95.6% of the isolates. Among the taxa identified,Alternaria alternata,Colletotrichum fioriniae,Colletotrichumgodetiae,Coniella quercicola,Diaporthe eres,Gnomoniopsis paraclavulata,Ilyonectria rufa,Mucor genevensis,Penicillium glandicola,Tubakia dryina, andUmbelopsis changbaiensiswere most frequently isolated, and were consistently found in stems and roots of both healthy and diseased plants. The community compositions of fungi in healthy and diseased stems were similar, althoughFusariumspecies, especiallyFusariumsp. 1 (FTSC 5) andF.sporotrichioides, were found mainly in seedlings with symptoms. The pathogenicity of the most consistently isolated species from stems and roots of diseased seedlings was tested onQ.roburseedlings.F.sporotrichioidescaused the largest lesions on inoculated seedlings. Six weeks after inoculation,D.eres,C.fioriniae,G.paraclavulata,T.dryina, andF.sporotrichioideskilled 0%–18.8% of seedlings, whileI.rufaandIlyonectriapseudodestructansdid not cause any lesions or other symptoms. This study is the first comprehensive report suggesting that massive fungal attack can lead to oak seedling dieback in mixed‐species forest stands in central Europe.