Sterile Potato Dextrose Agar Plugs Research Articles

First Report of Phytophthora inundata Associated with Decline and Death of Walnut (Juglans regia L.) in Italy.

Persian walnut (Juglans regia L.) is an important nut crop in Italy. In recent years, incidence of walnut decline and death has increased in many Italian commercial orchards. In early summer 2020, we observed a serious decline in approximately 5% of trees in a waterlogged area of a Veneto-region walnut orchard (J. regia, cv Lara). Symptoms included extensive foliar wilt and canopy decline associated with collar and root rot. Symptomatic tissues excised from larger roots of affected trees were surface disinfested for 1 min in a 1% NaOCl solution, rinsed for 5 min in sterile distilled water, and placed onto P5ARPH selective medium (Jeffers and Martin 1986). A Phytophthora-like organism was consistently isolated. Pure cultures were obtained by single-hyphal transfers onto potato dextrose agar (PDA). Isolates were identified as Phytophthora inundata based on morphological characteristics (Brasier et al. 2003), sequences of internal transcribed spacer (ITS) amplicons from universal primers ITS6 (Cooke et al. 2000) and ITS4 (White et al. 1990) and sequences of cytochrome c oxidase, subunit II (Cox II) from Fm75 and Fm78 primer pair (Martin and Tooley 2003). On carrot agar (CA), colonies had a characteristic stellate to broad lobed patterns. On this medium, optimal growth was at 28-30 °C (7,3 mm/day) and the upper temperature limit for mycelial growth was 37°C. Mycelial disks of isolate CREADC-Om306, grown on CA, were floated in Petri plates with soil extract solution and incubated under continuous fluorescent light at room temperature (25+/-2 °C). Within 48 to 72 h, sporangia were produced that were persistent, non-papillate, ovoid or ovoid-obpyriform, measuring 55.0 to 80.7 (length) x 41.3 to 65.2 (width) µm (averages 64.3+/-10.2 x 47.9+/-9.7 µm). Oospores and chlamydospores were absent. BLAST analysis of the amplicons from CREADC-Om306 revealed ITS sequences (854-bp; GenBank accession no. OK342200) and Cox II sequences (568-bp; GenBank accession no. OK349677) that shared 100% identity with published P. inundata sequences available in GenBank (acc. n. AF266791 for ITS; MT458994 for Cox II). Pathogenicity tests were conducted in the greenhouse on six 2-year-old walnut (J. regia, cv Lara) plants. Four of the plants were inoculated with CREADC-Om306 on two opposite sides of each plant's stem at 1-2 cm above soil line. A cork borer was used to remove a 5-mm disk of bark that was replaced by a 5-mm diameter mycelial plug from 10-day-old cultures of the pathogen on PDA. Two control plants were treated in the same way except the bark wounds were inoculated with sterile PDA plugs. Plants were kept in greenhouse at 24 ± 2°C. After 3 months, lesions had developed from all points of inoculation with. P. inundata (mean lesion length 55,25+/-6,22 mm) and the pathogen was reisolated from the lesion margins of all inoculated plants. The control plants remained symptomless and did not yield the pathogen. P. inundata is widely distributed across the world as a plant pathogen on several native as well as horticultural crops, especially in riparian or other areas subject to flooding or waterlogging. This report is the first to document P. inundata as a pathogen on Persian walnut and adds it to the diverse list of known susceptible perennial native, ornamental, and agricultural hosts of this organism. In addition to P. inundata, which belongs to the major Phytophthora ITS Clade 6, other members of the clade including P. megasperma (Belisario et al. 2012) and P. gonapodyides (Belisario et al. 2016) have been described as walnut pathogens.

First Report of Red Leaf Midrib lesions on Sugarcane Caused by Lasiodiplodia theobromae in China.

Sugarcane (Saccharum officinarum) is an economically important crop and is extensively planted across China. In August 2020, leaf midribs with red lesions were observed on cultivar 'Yunzhe 081609' in Kaiyuan (103.27°E, 23.71°N), Yunnan, Southwestern China. In July to August 2021, similar symptoms were observed on cultivar 'Liucheng 05-136' in Hechi (108.48°E, 24.47°N), Guangxi, and on cultivars 'Yingyu 91-59' and 'Yunzhe 081609' in Lingcang (99.45°E, 23.33°N), Yunnan. Initially symptoms appeared as red spots on the leaf midribs, which gradually expanded, forming elongated red lesions. At high severity, the leaves broke and hung down. Disease incidence of leaves was estimated at 30 to 50% across the locations. To identify the etiology of this disease, three symptomatic leaves were collected from cultivars 'Liucheng 05-136', 'Yingyu 91-59', and 'Yunzhe 081609', respectively. Symptomatic leaf midribs were cut to small fragments (3 × 5 mm), surface sterilized with 70% ethanol for 30 s followed by 1% NaClO for 1 min, rinsed with sterilized distilled water three times, air dried on sterile filter paper, plated on potato dextrose agar (PDA), and incubated at 28°C in the dark. Ten isolates with similar morphological characteristics were obtained. Colonies on PDA were white to grayish-white with aerial mycelium growing initially upward and then forming clusters. After 10 days, mycelia turned to grayish black. Immature conidia were initially hyaline, aseptate, and ellipsoid. Mature conidia became dark brown, septate, longitudinal striate, and measured 21.2 to 25.8 × 11.4 to 16.4 µm (n = 30). Morphologically, the isolates were identified as Lasiodiplodia theobromae (Alves et al. 2008). For molecular identification, genomic DNA of four representative isolates (LTGX1, LTGX2, LTYN1 and LTYN2) was extracted using the Ezup Column Fungi Genomic DNA Purification kit. The internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-alpha (TEF-1α) gene, and β-tubulin (TUB) gene were amplified with primer pairs ITS1/ITS4 for ITS, EF1-728F/EF1-986R for TEF-1α, and Bt2a/Bt2b for TUB, respectively (Glass and Donaldson 1995; Carbone and Kohn 1999; White et al. 1990), and then sequenced. The ITS (ON533336-ON533339), TEF-1α (ON939550-ON939553) and TUB (OP747306-OP747309) sequences were deposited in GenBank. BLAST searches showed >99% nucleotide identity to the sequences of ex-type isolate CBS 164.96 of L. theobromae (ITS, 99.8% to AY640255; TEF-1α, 99.9% to AY640258; TBU, 100% to EU673110). Phylogenetic analysis using maximum likelihood based on the combined ITS, TEF-1α, and TUB sequences of the isolates and reference sequences of Lasiodiplodia spp. downloaded from the GenBank indicated the isolates obtained in this study formed a clade strongly supported based on bootstrap values (100%) to the ex-type isolate CBS 164.96 sequences of L. theobromae. For pathogenicity tests, three healthy 6-month-old potted sugarcane leaf midribs of cultivar 'Yunzhe 081609' were wounded with a sterile needle, then inoculated using 8-mm mycelial agar plugs from a 10-day-old culture of strain LTYN1, and covered with wet cotton to maintain high relative humidity. Sterile PDA plugs were used as controls. Plants were placed in a greenhouse at 28 to 32°C. The test was conducted twice. Five days after inoculation, red lesions appeared on the inoculated leaf midribs. These symptoms were similar to those observed in the field. The leaves used for negative controls remained symptomless. The same fungus (L. theobromae) was re-isolated from all inoculated-symptomatic tissues; and isolates had the same morphological traits mentioned above. The DNA sequence data of these isolates was also similar than the original isolates. The association of L. theobromae with S. officinarum was recorded earlier in Cuba (Urtiaga, 1986), Myanmar (Thaung, 2008) and the Philippines (Reinking, 1919). Leaf midribs with red lesions caused by Colletotrichum falcatum has already been described around the world (Costa et al. 2021; Hossain et al. 2021; Xie et al. 2019). All together, this information indicates that L. theobromae is one of the causal agent of the red lesions symptoms on the sugarcane leaf midribs. To our knowledge, this is the first report of L. theobromae causing red lesions on leaf midribs of sugarcane in China. Further research will focus on developing management strategies to control this disease effectively.

First report of leaf spot and stem blight on blueberry (Vaccinium corymbosum cv. Bluerain) caused by Calonectria pseudoreteaudii in China.

Blueberry has high nutritional value and is one of the five healthy fruits. In 2018, leaf spots and stem blights were observed on Vaccinium corymbosum cv. Bluerain in Guangzhou, Guangdong Province, China. Up to 80% of the plants were affected. Initial symptoms of affected leaves were red-brown, irregular, small spots, which gradually coalesced and formed larger irregular necrotic patches. The affected stems showed red-brown and irregular large lesions. Diseased tissues were surface sterilized with 75% alcohol for 15 s, followed by 2.5% NaClO for 30 s, and rinsing three times in sterile distilled water, placed on potato dextrose agar (PDA) and incubated at 25 C. Representative strains, ZHKUCC 21-0021 from diseased leaves and ZHKUCC 21-0073 from diseased stems, were selected for further studies. Colonies grew slowly at 25 C on malt extract agar (MEA) (average 5.68 mm/d), producing white aerial mycelium and red-brown color on the underside after 7 days. Macroconidiophores were hyaline, smooth, consisting of a stipe bearing fertile branches, and a stipe extension terminating in a vesicle. Each terminal branch produced 2-4 phialides, 8-13 × 3-6 μm, reniform or doliiform; Stipe extensions were septate, terminating in a narrowly clavate vesicle, 2-6 μm. Macroconidia were hyaline, straight cylindrical, round at both ends, 83-100 × 7-11 μm (average = 94 × 8 μm; n = 50), with 5 septa. These morphological characteristics were similar to the description of Calonectria pseudoreteaudii (Lombard et al., 2010). The partial calmodulin (cmdA), beta-tubulin (β-tubulin), and translation elongation factor 1-alpha (tef1-α) genes of the two isolates were respectively amplified using primers CAL-228F/CAL-737R (Carbone et al., 1999), EF1-728F/EF2 and T1/CYLTUB1R (Lombard et al., 2015), and sequences were deposited in GenBank (cmdA: MZ516854 and MZ516855; β-tubulin: MZ516858 and MZ516859; tef1-α: MZ516856 and MZ516857). BLAST analysis of three gene sequences showed 100% similarity to those of C. pseudoreteaudii. In the maximum likelihood (ML) tree of the concatenated sequences of the three genes, the two isolates from this study were clustered with C. pseudoreteaudii with 100% bootstrap support. Five-mm-diameter hyphal plugs of two representative isolates grown on PDA for five days were used in the pathogenicity test. Leaves were inoculated with ZHKUCC 21-0021, and stems were inoculated with ZHKUCC 21-0073 with five replicates. As controls, sterile PDA plugs were used. All inoculated plants were maintained at 25 C . After 7 days, inoculated leaves and stems developed symptoms similar to field samples, whereas the control plants remained asymptomatic. The pathogen was reisolated from inoculated plants and confirmed to be C. pseudoreteaudii by morphological characteristics. Five Calonectria species (C. canadensis, C. colhounii, C. ilicicola, C. kyotensis and C. pyrochroa), have been reported associated with blueberry (Farr and Rossman, 2022; Fei et al, 2017). Calonectria canadensis and C. ilicicola have been reported to cause stem blight and stem rot in Vaccinium spp. in China (Fei et al, 2017 and 2018). Calonectria colhounii has been reported to cause stem blight in V. angustifolium and V. corymbosum in the United States (Sadowsky et al, 2011). However, this is the first report of C. pseudoreteaudii causing leaf spot and stem blight on Vaccinium spp. worldwide. These results will provide a foundation for future research on prevention and control of this disease.

First report of Phytophthora blight caused by Phytophthora nicotianae on Daphne odora in China.

The variegated leaves and fragrant flowers of Daphne odora var. marginata Mak. make it a popular garden plant. In May 2020, we found diseased D. odora plants in a greenhouse at the Ganzhou Vegetable and Flower Research Institute, in southeast China; 72% of 1800 plants had Phytophthora blight-like symptoms-shrunken stems, black withered branches, wilted and dropped leaves (Fig 1a), and rotted and dark green roots. The root and stem tissue surfaces were disinfected with 75% ethanol for 30s followed by 0.1% HgCl2 for 1min, rinsed thrice with sterile water, and cultured on potato-dextrose agar (PDA) medium at 25°C. Mycelia from the diseased tissue were subcultured on fresh PDA medium, providing three colonies. White colonies (~4.1mm) were formed after 10 days at 25°C (Fig 1b). Sporangia and chlamydospores were induced by placing actively growing mycelia on PDA medium at 25°C for ~30 days and then at 45°C for ~3 days. Sporangia were ovoid to spherical and 19.33 × 20.99µm in size (Fig 1c), whereas chlamydospores were spherical and 15.68 × 16.10µm in size (Fig 1d). All three colonies resembled Phytophthora spp. Genomic DNA was extracted from isolates using the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech [Shanghai] Co. Ltd.), and rDNA-ITS and β-tubulin were amplified and sequenced. BLAST analysis (GenBank) revealed that the ITS (Accession No. MZ676071) and β-tubulin (MZ748503) sequences of isolates shared the highest similarity (99-100%) with those of Phytophthora nicotianae (Duccio et al. 2015). A phylogenetic tree of the relationship between our isolate hjt3 and its close relatives within the P. nicotianae species was constructed using the MEGA X neighbor-joining method (Fig 2). The pathogen was identified as P. nicotianae based on morphological and molecular characteristics. Sequencing results of the three samples were consistent, all indicating P. nicotianae. A specimen (JXAU-H2020245) was deposited in the Herbarium of the College of Agronomy, Jiangxi Agricultural University. To confirm pathogenicity, 9-month-old healthy D. odora plants were used for stem and soil inoculation. Stems were cut ~5 cm from the soil with sterilized scalpels and inoculated with 0.8 cm diameter PDA plugs containing actively growing mycelia of isolate hjt3. The soil was sterilized and 0.8 cm PDA plugs containing actively growing mycelia were buried in the soil at ~5cm; the mycelia were in contact with the roots. Plants in both groups were treated equally; those inoculated with sterile PDA plugs served as controls. There were six plants in each group, with each experiment performed in triplicate. All plants were incubated in a greenhouse at 25-28°C. The stems shrank and began to rot rapidly after 7 days (Fig 3) and the branches turned black and withered within 2 weeks. After soil inoculation, the stems of the inoculated plants blackened and rotted in ~20 days (Fig 4) and the roots rotted and turned dark green (Fig 5). These symptoms rapidly spread to the branches. The control plants did not exhibit any symptoms. Reisolated colonies showed the same morphological traits as the isolates used for inoculation; no target colonies were isolated from the control plants. Phytophthora blight caused by P. nicotianae on D. odora has been reported in Italy (Garibaldi A, 2009) and Korea (Kwon et al. 2005). This is the first detection in China. Therefore, Phytophthora blight on D. odora caused by P. nicotianae should be monitored and controlled to promote the development of the D. odora industry.

First report of Taxus media branch blight caused by Neopestalotiopsis clavispora in China.

Taxus media Rehder is a high-value tree species and can be used for landscape afforestation and medicinal purposes considering its beautiful shape and secondary metabolite paclitaxel. In May 2019, branch blight of T. media was observed in plantations in Hangzhou, Zhejiang province, China. The lesions were brown, necrotic and sunken, and the branches were withering. Plants from five plantations were surveyed, and the disease incidence was 60%. Necrotic tissues from 5 plans from each plantation were cut into small pieces (5×5 mm), surface disinfected with 75% alcohol for 30 s and 2% sodium hypochlorite solution for 2-3 min, washed twice with sterile water, then dried on sterile filter paper. Branch pieces were aseptically transferred to potato dextrose agar (PDA) plates and incubated at 25℃ in the dark for 3 days. Five purified fungal cultures were obtained and the representative isolate (LA-01) that grew slightly more vigorously under the same condition was selected for morphological characterization and DNA sequence comparison. The colony was initially white and cottony, with circular growth, undulated edge, wavy surface and pale beige on the back. The conidia were long spindle-shaped, straight or slightly curved, 21-27 × 6-8 μm (n=50) in overall shape. They contained 5 cells with those at the apex and base hyaline. The second and third cells from the apex were darker (brown) than the fourth cell from the apex (pale brown). Two or three (most frequently observed) hyaline appendages were apparent at the apex, 16-31 μm in length. Morphological characteristics of the obtained isolate were consistent with those in the genus of Neopestalotiopsis (Solart et al. 2018). DNA of LA-01 was extracted from fresh mycelia using Minibest Universal Genomic DNA Extraction Kit (Takara, Toyoto, Japan) according to the manufacturer's instructions. The partial regions from the internal transcribed spacer (ITS), β-tubulin gene (TUB2), and translation elongation factor subunit 1-a gene (TEF1) were amplified and sequenced using universal primer pairs ITS5/ITS4, BT2A/BT2B, and EF1-526F/EF1-1567R (Maharachchikumbura et al. 2012), respectively. The resulting nucleotide sequences were individually subjected to BLAST searches in GenBank. The nucleotide sequences of ITS (MZ148633, 554 bp), TUB2 (MZ286974, 473 bp ), TEF1 (MZ286975, 1030 bp) of the isolate LA-01 showed 99.64% similarity to ITS sequence (MW512858.1), 99.35% similarity to TUB2 sequence (MH423961.1), 98.18% similarity to TEF1 sequence (KU096881.1). The multi-locus phylogenetic tree was constructed based on the combined ITS-TUB2-TEF1 sequences using MEGA 7 using the neighbor-joining method, indicating that LA-01 represents Neopestalotiopsis clavispora. Inoculation was done on branches of ten T. media plants in the field to verify the pathogenicity of LA-01. Two healthy branches of each plant were surface sterilized with 75% ethanol allowed to dry, and two small wounds on each branch were obtained by puncturing with a sterilized needle. One wound was inoculated with 5-mm-diameter PDA discs excised from 7-day-old cultures, and wrapped with parafilm; the other inoculated with sterile PDA plugs as control, and wrapped with parafilm. Disease symptoms developed on all N. clavispora-inoculated wounds 3 days after inoculation. The yellow brown lesions became apparent 20 days later, whereas the control wounds remained asymptomatic. The fungus N. clavispora was reisolated from the diseased lesions using the same methods above, fulfilling Koch's postulates. Previously, the species was reported to cause leaf spot disease on another Taxus tree species T. chinensis in China (Wang et al. 2019). This is the first report of T. media branch blight caused by N. clavispora worldwide. Special attention should be paid to the damage it may cause to T. media producers, and precautionary monitoring on T. media population will be a significant part of the conservation efforts in China.

First Report of Alternaria alternata Causing Leaf Spot on Bellis perennis in China.

Bellis perennis L., commonly known as the daisy or sun chrysanthemum, belonging to the family Asteraceae, is a perennial herb and is usually used as an ornamental plant worldwide for its vibrant flowers. Simultaneously, B. perennis has been proved to have therapeutic effects used on common colds, wound healing, anti-tumor, anxiolytic and antioxidant (Karakas et al. 2017). In July 2021, typical leaf spot was observed on B. perennis with about 50% disease incidence in Ruyue lake wetland park of Zibo (36.71°N, 118.01°E), Shandong Province, China. We surveyed more than 1000 square meters of planting area, and the diseased leaves were mostly concentrated in the lower location of plants, where the humidity was higher under the forest. Symptoms on the initially diseased leaves appeared as light yellow, round or oval lesions with light or brown borders. With the development of the disease, the area of the lesion gradually expands, the color deepens, and the shape is becoming irregular. To identify the causal pathogen, small pieces of 15 tissues collected from the infected leaves were sterilized with 75% ethanol for 30 s and then 2% sodium hypochlorite (NaClO) for 60 s, finally rinsed with sterile water three times. All the tissues were placed on potato dextrose agar (PDA) and incubated at 25 ℃ in the dark for 5 days (Zhu et al. 2013). A total of 13 isolates were obtained from the above diseased leaves. The cultures were initially grayish white, then a light green halo appeared in the middle of the medium after 5 days, with numerous gray aerial hyphae. For molecular identification, the RNA polymerase II beta subunit (PRB2), Tsr ribosome biogenesis protein, partial coding sequences of chitin synthase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and major allergen Alt a 1 were amplified from genomic DNA extracted from four representative single isolates using the primers PRB2DF/PRB2DR, Tsr1F/Tsr1R, CHSDF1/CHSDR1, GDF1/GDR1, and AltF/AltR (Damn et al. 2019; Lawrence et al. 2013), respectively, and sequenced (GenBank accession nos. OL416000, OL416001, OL416002, OL416003, and OL416004). These genes had more than 99.9% nucleotide identity with the corresponding sequences (KY131957.1, KY131958.1, KY996470.1, MN657411.1, and KY923227.1) of the reference strains of Alternaria alternata in GenBank. For pathogenicity tests, five healthy B. perennis plants each with three living leaves were inoculated with mycelial plugs of A. alternata from a 5-day-old culture grown on PDA. After inoculation, the plants were placed in a greenhouse with 85% relative humidity at 25 ℃ and monitored daily for symptom development. After 3 days, all inoculated leaves with mycelial plugs of A. alternata appeared symptoms similar to those observed in the field previously, while no symptoms appeared on negative controls which were inoculated with sterile PDA plugs. Cultures re-isolated from diseased leaves had the same morphological and molecular results as those isolated in the field, confirming Koch's postulates. The causal agent on B. perennis was confirmed as A. alternata on the basis of morphological and molecular results (Simmons 2007). To our knowledge, this is the first report on the presence of A. alternata affecting B. perennis plants in China. The discovery of this new disease is beneficial to the application and protection of B. perennis, which is a popular landscape and medicinal plant.

First Report of Brown Rot Caused by Monilinia polystroma on Quince in Italy

HomePlant DiseaseVol. 107, No. 1First Report of Brown Rot Caused by Monilinia polystroma on Quince in Italy PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Brown Rot Caused by Monilinia polystroma on Quince in ItalyU. Spitaler, S. Oettl, and E. DeltedescoU. Spitaler†Corresponding author: U. Spitaler; E-mail Address: urban.spitaler@laimburg.ithttps://orcid.org/0000-0003-0492-6129Institute for Plant Health, Laimburg Research Centre, Auer (Ora) 39040, South Tyrol, ItalySearch for more papers by this author, S. OettlInstitute for Plant Health, Laimburg Research Centre, Auer (Ora) 39040, South Tyrol, ItalySearch for more papers by this author, and E. Deltedescohttps://orcid.org/0000-0002-0697-3642Institute for Plant Health, Laimburg Research Centre, Auer (Ora) 39040, South Tyrol, ItalySearch for more papers by this authorAffiliationsAuthors and Affiliations U. Spitaler † S. Oettl E. Deltedesco Institute for Plant Health, Laimburg Research Centre, Auer (Ora) 39040, South Tyrol, Italy Published Online:24 Oct 2022https://doi.org/10.1094/PDIS-06-22-1442-PDNAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Brown rot, caused by Monilinia spp., is an important disease of stone and pome fruits in Italy. The main pathogens are Monilinia laxa, M. fructigena, and M. fructicola. In 2009, a new species, Monilinia polystroma (G. Leeuwen) L. M. Kohn, was found in Europe (Petróczy and Palkovics 2009). The first reports of M. polystroma on pome fruit for Italy were on pear in 2013 (Martini et al. 2015) and on apple in 2021 (Rosati et al. 2021). During a survey in Italy, it was observed that quince (Cydonia oblonga Mill.), a plant of the Rosaceae family and a pome fruit, was frequently affected by brown rot. To determine the pathogen, single spore isolates were obtained from symptomatic fruits of a quince plant in Kaltern/Caldaro (South Tyrol, Italy) in September 2021. On the selected tree, one-third of the fruit showed brown rot symptoms. The isolates showed morphological characteristics compared to M. polystroma on potato dextrose agar (PDA) at 22°C and a photoperiod of 16 h (Vasić et al. 2016). The colonies were grayish brown with slightly undulate margins. The colony reverse developed black stromatal plates in concentric rings within 14 days post inoculation (dpi). DNA was extracted from mycelium and the internal transcribed spacer (ITS) region using ITS6/ITS4 primers (White et al. 1990) as well as a genomic sequence with unknown function using the primers UniMon_Forw/UniMon_Rev (Petróczy et al. 2012), were amplified and sequenced. MegaBLAST analysis revealed 100% identity with M. polystroma (accession nos. NR_154198 and JN128836, respectively). Sequences were deposited in NCBI GenBank under the accession numbers ON646524 and ON806527. To confirm pathogenicity, 10 mature pear quinces from organic production were surface sterilized with 75% ethanol. Mycelial plugs (2 mm) were dislodged from an 8-day-old colony and inserted into a 3 mm hole in the quince. Ten fruits inoculated with sterile PDA plugs were used as a control. Quinces were incubated in a plastic box at 22°C, 16 h photoperiod, and 100% relative humidity. Brown rot lesions developed within 1 day. Additionally, inoculation with spores was performed. One-celled, limoniform, hyaline conidia, measuring 11.7 to 19.4 × 8.8 to 13.1 μm, were obtained from mycelium-inoculated quinces 9 dpi. Ten quinces were wounded with a sterile needle and inoculated with 20 μl conidial suspension (1.1 × 104 spores/ml). Ten quinces inoculated with sterile water were used as a control. After 4 days, brown rot lesions developed on all spore-inoculated fruits. Mean lesion radius (±SD) was 2.5 (±0.8) mm 4 dpi, 25.4 (±1.5) mm 7 dpi, and 45.0 (±4.4) mm 11 dpi. One month after inoculation, all quinces were completely covered by the lesion, while all control fruits remained symptomless. To confirm identity, the fungus was isolated from four spore-inoculated fruit. The isolates showed the same morphological and molecular features as the original isolate. Sequences were deposited in GenBank under the accession numbers ON646525 to ON646528 and ON806528 to ON806531. Although the presence of M. polystroma has been documented for Italy, this is the first report of this species on Italian quinces. The high number of infected fruits found in this study suggests a broad impact of M. polystroma on Italian quince cultivation. Due to the widespread cultivation of quinces, additional knowledge of the occurring pathogens will help to assign brown rot symptoms to M. polystroma and to improve targeted control strategies.The author(s) declare no conflict of interest.

The first report of Lasiodiplodia theobromae causing leaf spot on Illicium verum in China.

Star anise (Illicium verum) production is an important industry in parts of southern China (Wang et al. 2011). The production of star anise (cultivar Dahong) was seriously affected by a leaf spot disease during the summer of 2020 in Rong County, Guangxi province, China. Approximately 20% to 30% of the trees (n = 200, 15-16 years old) had obvious leaf spots on more than 80% of the leaves. Local growers had to cut down the seriously diseased trees. Symptomatic samples were collected and sent to us at the end of August 2021. A single brown-edged round gray spot appeared on each leaf. The spot was ~20 mm in diameter. The margin was cut into 5 mm pieces, then disinfested with 3% NaOCl for 30 s, 75% ethanol for 30 s and sterile deionized water for 1 min before dried and placed on potato dextrose agar (PDA) medium at 25°C in the dark. A total of 25 fungal isolates were obtained (isolation rate 82%). Genomic DNAs was extracted from the mycelia of these isolates and three diagnostic regions including ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and Bt2a/Bt2b (Glass and Donaldson 1995) were amplified. The colony morphology on PDA and the sequences of the five isolates BJ20-1, BJ20-4, BJ20-5, BJ20-7, BJ20-8 were identical. Fungal colonies had light gray mycelium and black pigment on PDA. The average colony growth rate was 4.25±0.31 cm per day and no spores were produced. Sequences of the representative isolate BJ20-1 were deposited in GenBank (Accession nos. OK483326, OL547596 and OL547597). BLASTn search indicated high identity 99.6%, 98.58% and 100% to ITS (AY640255), EF1-α (AY640258), and β-tubulin (KU887532) of Lasiodiplodia theobromae, respectively. Combined phylogenetic analysis using MEGAX (Kumar et al. 2018) clustered BJ20-1 and L. theobromae CBS164.96 in one clade. To test pathogenicity, 2-years-old healthy I. verum trees (cultivar Dahong) maintained in a greenhouse were inoculated. Leaves were surface sterilized with 70% ethanol. One PDA plug (5mm in diameter) was placed on each wound acupunctured with a sterile needle pick. Ten PDA plugs with mycelial of BJ20-1 growing 7 days on it were inoculated on five leaves. Four sterile PDA plugs placed on two leaves served as controls. All the plugs were removed from the leaves the day after inoculation. The experiment was repeated three times. At 1 day post-inoculation (DPI), brown expanding lesions were observed on the inoculated leaves. At 7 DPI, a mature ellipse of necrosis formed with 18±4 mm in diameter with black pycnidia in the center. Conidia were observed in the pycnidia. The immature conidia were thick-walled, hyaline, aseptate and ellipsoid, measuring 20 - 25.2 × 11 - 13 μm (n = 25). The mature conidia were dark brown with one central septum, measuring 24.3 - 27 × 13 - 14 μm (n = 25). At 10 DPI, the control leaves remained asymptomatic. Re-isolation was successful from the spot on the inoculated leaves. The colony morphology and molecular identification of the re-isolations were all the same as that of BJ20-1. In conclusion, the morphological and molecular evidence consistently indicated these isolates were L. theobromae. Koch's postulates were fulfilled that L. theobromae was pathogenic on star anise. Although L. theobromae has been reported to cause leaf spot disease on Camellia sinensis (Bao et al, 2021), Kadsura longipedunculata (Fan et al, 2020) and Broussonetia papyrifera (Luo et al, 2020), etc., this is the first report of L. theobromae causing leaf spot on I. verum in China. Due to the leaf spot disease resulting in serious yield reduction on star anise, accurate pathogen identification in this study would significantly improve the control of the leaf spot disease on star anise.

First Report of Crown Rot Caused by Fusarium algeriense on Wheat in Kyrgyzstan.

Fusarium crown rot of wheat is an economically important disease that leads to significant yield and quality losses, especially in many arid and semi-arid wheat-growing areas worldwide. In June 2020, winter wheat (Triticum aestivum L.) plants exhibiting crown rot symptoms were identified in a commercial field located in the Tokbay location (43.033719°N, 74.325623°E), Chuy Province, Kyrgyzstan. The diseased plants were stunted and had brown discoloration on internodes of the stem bases and roots. Disease incidence was about 3%. A total of 10 plants were sampled at the ripening stage from the field to identify the causal agent. Symptomatic tissues were excised, surface disinfected with 1% NaOCl, rinsed three times with distilled water, and placed on one-fifth strength potato dextrose agar (PDA) followed by incubation at 23°C in the dark for 5 days. A total of 8 Fusarium isolates were recovered from tissues and purified by the hyphal tips method onto fresh PDA and Spezieller-Nährstoffarmer agar (SNA) plates (Leslie and Summerell 2006). Sequence analysis of the translation elongation factor 1α (TEF1) and the RNA polymerase II beta subunit (RPB2) genes were performed with primers EF1 and EF2 (O'Donnell et al. 1998), and 5f2 (Reeb et al. 2004) and 7cr (Liu et al. 1999), respectively. The sequences of three isolates showed 100% identities with the corresponding sequences of the strain NRRL 66652 of Fusarium algeriense Laraba & O'Donnell (TEF1: MF120515 and RPB2: MF120504), and the sequences of a representative isolate (KyrFa01) were deposited in GenBank (TEF1: OM135603 and RPB2: OM135604). On PDA, fungal colonies were initially yellowish-white but gradually turned yellowish-brown. Ellipsoidal microconidia produced in false heads on monophialides were usually aseptate (8.30 ± 1.17 μm, n = 50) and occasionally one-septate (21.89 ± 2.01 μm, n = 50). Sporodochial macroconidia were mostly 3-4 septate measuring 43.41 ± 2.83 μm (n = 50), slightly curved and formed generally on monophialides on SNA. No chlamydospores formation was detected after 15 days on SNA or PDA. Morphological characteristics described above were consistent with the morphology of F. algeriense, as reported by Laraba et al. (2017). To confirm pathogenicity, seeds of wheat cultivar Seri 82, Fusarium crown rot susceptible, were treated in 1% NaOCl for 2 min, rinsed twice, and placed in plates containing a piece of sterile filter paper saturated with water to induce germination for 3 days. Five pregerminated seeds were placed on the soil surface for each 9-cm-diameter pot, which was filled with a sterile potting mix containing peat, vermiculite, and soil (1:1:1 by v/v/v). A 1-cm-diameter mycelial plug taken from the margin of actively growing colonies (PDA) of the representative isolate KyrFa01 was contacted with each seed, and then seeds were covered with the same potting mix. The seeds in control pots were treated with sterile PDA plugs. The experiment was conducted in a growth chamber in a completely randomized design with five replicated pots at 23°C with a 12-h photoperiod. Disease assessment was made after 4 weeks of fungal inoculation. The isolate KyrFa01 induced discoloration on the crown and root tissues of inoculated plants similar to those observed in the field-grown plants, whereas no symptoms were observed on plants grown in the control pots. The pathogen was successfully reisolated from the symptomatic tissues, confirming Koch's postulates. To the best of our knowledge, this is the first report of crown rot caused by F. algeriense on wheat in Kyrgyzstan. Fusarium algeriense was firstly described within the Fusarium burgessii species complex by Laraba et al. (2017) as a crown rot pathogen of wheat in Algeria. The pathogen was secondly reported from wheat-growing areas in Azerbaijan (Özer et al. 2020a) and thirdly from Kyrgyzstan in this report. Özer et al. (2020b) confirmed the coexistence of this pathogen with other Fusarium species. The result warrants the need to further investigate the potential of this species in the Fusarium crown rot complex of wheat.

First report of Coniella granati causing leaf spot of pomegranate (Punica granatum L.) in Hungary.

Pomegranate (Punica granatum L.), the hystoric fruit and ornamental crop native to Iran and North India is widely planted in the Mediterranean and became popular in the house gardens of northest parts of Europe (Fernandez et al. 2014) including Hungary. In August 2020 necrotic black lesions and serious defoliation were observed on 60% of 1-3 year old pomegranate trees (cv. Wonderful) in a horticultural nursery near Gödöllő, Hungary (47°36'00.9"N 19°21'26.5"E). Symptoms started as small irregular dark brown spots on the leaves, which later increased in size (2.6 ± 0.9 mm). Ultimately, the entire leaf turned yellow, defoliation resulted in damage on (6) - 8 - (15)% of the leaves. Then, black pycnidia with unicelled, elliptical to fusiform, colourless conidia (Avg. 50 conidia: 2.4 - (3.6) - 3.9 × 10.2 - (13,1) - 17.9 µm) developed on the surface. These morphological features matched those described earlier by Van Niekerk et al. (2004) and Alvarez et al. (2016) for C. granati. Conidia from pycnidia were directly transferred to potato dextrose agar (PDA) by sterile needle. The plates were incubated at 24°C in the dark. Light yellow colonies with whitish aerial mycelia and later black globose pycnidia were observed. Mass of conidia oozed from pycnidia after 15 days of incubation. Pathogenicity tests were carried out on 1-year-old potted P. granatum trees (cv. Wonderful) with 5 replicates in the greenhouse. Ten, randomly selected leaves were inoculated per plant. 7-mm mycelial plugs from the edge of 10-day-old colonies were placed directly on disinfested (2% NaOCl solution, than sterile distilled water) leaves. The plants were covered with plastic film for 3 days after inoculation (26±3°C and 87±3% relative humidity). Pathogenicity was also tested on nonwounded, surface-disinfested fruits by mycelial plugs in 3 × 3 replicates. Inoculated fruits were placed in large grass vessels for 15 days (24±2°C and 80±5% relative humidity). Uncolonized, sterile PDA plugs were used as controls in both cases. Dark brown legions developed after 9-12 days on the plants in the greenhouse. On pomegranate fruits, the fungus colonized the fruit after 7-8 days, followed by fruit rot. In some cases, after 2 weeks pycnidia developed on the skin surface. No decay were present on control leaves or fruits. The pathogen was reisolated from all infected tissues and identified as C. granati, thus fulfilling Koch's postulates. For molecular identification, total genomic DNA of the isolate was extracted from the growing margins of colonies on PDA and partial sequence of internal transcribed spacer (ITS) and translation elongation factor 1-alpha (tef1) were amplified by PCR using primers described by Alvarez et al. (2016). Sequence data of the Hungarian isolate of the ITS region (GenBank acc. no. MW581953) showed 99.8% identity (559 bp out of 560 bp) with C. granati sequences deposited in GeneBank (Acc. nos. MH860368, MH855389 and KX833582). Considering tef1 sequence of the Hungarian isolate (OM908764) obtained had complete identity with other published C. granati isolates (KX833676, KX833682). C. granati has been previously reported on pomegranate from Europe (Palou et al. 2010, Pollastro et al. 2016). Based on morphological and molecular studies, this is the first record of C. granati in Hungary. The economic importance of this disease in currently limited in Hungary due to pomegranate is rather an ornamental crop, however, the first cultivation trials have been already started. There is a risk that the spread of the pathogen began with the infected propagating material, as a result the disease may outbreak anywhere in the country.

First Report of Sclerotinia minor on lettuce in Serbia.

Lettuce (Lactuca sativa L.) is the world's most popular leafy salad vegetable. One of the major challenges facing lettuce producers are fungal diseases that could, under favorable conditions, devastate the harvest (Raid, 2004). During February 2021, poor growth, plant stunning and blanching of leaves of lettuce plants, cultivated in unheated plastic tunel in Potočanje (Zlatibor region), Serbia, were observed. The crowns were softened with spreading decaying lesions covered with white mycelium, particulary on the leaves near the soil surface. Approximately 2 to 3 weeks before harvest, diseased plants began to wilt and collapse. Estimated disease incidence was 50-55%. In order to identify the causal agent, symptomatic tissues from diseased plants were cut into small pieces, surface sterilized with 70% ethanol for 1 min, rinsed three times in sterile distilled water and placed on potato dextrose agar (PDA). Five isolates with uniform morphology were derived from infected tissue. The colonies had fast-growing, white, cottony aerial mycelium, producing profuse numbers (184 sclerotia/ Petri plate in average) of small, black, irregularly shaped sclerotia, less than 2 mm in diameter. Based on morfological features, the isolates were identified as Sclerotinia minor Jagger (Kohn, 1979). To confirm the species identity, the internal transcribed spacer region of nuclear rDNA of a representative isolate 15-2 was amplified using the primer pair ITS1/ITS4 (White et al. 1990). Sequence analysis of ITS region revealed 100% nucleotide identity between the isolate 15-2 (GenBank Accession No. OL423632.1) and 14 isolates of S. minor from different parts of the world (e.g., accession Nos. MK356551.1, KY707828.1, JF2798801.1). Pathogenicity tests were conducted by artificial inoculation of 55-day-old lettuce plants cv. 'Majska kraljica', grown on commercial growth substrate in l L pots. The obtained isolates were grown on PDA for 7 days and mycelial plugs, 5 mm in diameter, were cut from the margin of the colony and placed mycelium-side down on undamaged ground-level leaves of lettuce plants. Two plugs per isolate were placed onto five plants each for a total of ten replicates per isolate. Negative control plants (5) were inoculated similarly with sterile PDA plugs. Inoculated plants were covered with transparent plastic bags, sprayed with water (under the plastic) twice a day for 3 to 5 days to maintain high humidity, and kept in a growth chamber at 22°C (13 h light). After 7 to 10 days, all pathogen-inoculated plants developed lettuce drop disease symptoms, whereas the control plants remained symptomless. The pathogen was reisolated from symptomatic leaves and Koch's postulates were completed by confirming the identity of the isolates. To our knowledge, this is the first report of S. minor on lettuce in Serbia. More research is needed to better understand this disease, establish control strategies and minimize the spread of the pathogen to other lettuce-producing areas of the country.

First Report of Southern Blight on Aloe vera Caused by Athelia rolfsii in China.

Aloe vera (L.) Burm f., which belongs to the family Aloaceae, is a perennial succulent plant and cultivated for its medicinal, cosmetic, vegetable and ornamental uses. In summer of 2021, about 15% (60 infected among 400 surveyed plants) of A. vera (A. barbadensis) plants in two gardens in Lishui, Zhejiang Province of China showed symptoms of southern blight disease. Symptomatic plants primarily exhibited slightly sunken water-soaked, dark brown lesions on taproot and basal part of the stems. As the disease progressed, leaves in the basal part of stems and subsequently the whole plant rotted and withered, with white mycelial mats occurring on infected stems and leaves. Numerous brown, spherical sclerotia were observed on the colonized tissues and soil surfaces around the infected plants. Mycelial fragments and sclerotia from symptomatic leaves were plated directly to potato dextrose agar (PDA) amended with 100 μg/ml streptomycin and incubated at 26°C in the dark. By hyphal-tip method, a total of five pure isolates were obtained from five diseased leaf samples. When cultured on PDA at 26°C for three days, colonies showed white and thick aerial mycelium, with a radial growth rate of 23.7 mm/day. Typical clamp connection structures were observed microscopically after three days and numerous globoid, rapeseed shape sclerotia, measuring 1 to 2 mm in diameter (n=50) formed after six days. These sclerotia were initially white and gradually turned dark brown with age. On the basis of morphological characteristics, the fungal isolates were identified as Athelia rolfsii (Curzi) C.C. Tu & Kimbr (anamorph Sclerotium rolfsii Sacc) (Mordue 1974). The internal transcribed spacer (ITS) and translation elongation factor 1-α gene (TEF1) regions of a representative isolate LHBJ2-4 were amplified and sequenced using the primers ITS4/ITS5 (White et al. 1990) and EF1/EF2, respectively (accession no. MZ956758 and OL365370). BLASTn search showed that the amplified ITS and TEF1 sequences had 99.71% (680/682 bp) and 99.80% (498/499 bp) identity with the A. rolfsii isolates CBS 115.22 (MH854711.1) and Sr_286 (JF267815), respectively. Neighbor-joining phylogenetic tree based on the ITS sequences revealed that LHBJ2-4 clustered with A. rolfsii isolates. For pathogenicity test, three potted A. vera plants (~30 cm tall) were inoculated by placing a 0.5 cm mycelial plug of isolate LHBJ2-4 (three-day old) at the base of each A. vera plant. Three A. vera plants inoculated with sterile PDA plugs served as controls. All the inoculated plants were placed in a growth chamber at 27°C under a 12/12 h light/dark cycle. The inoculation assays were carried out twice. After 5 to 7 days, stem bases of the inoculated plants showed brown lesions that were similar to those observed in the field. However, control plants remained symptomless. Athelia rolfsii was re-isolated from all the inoculated plants and identified using morphological and molecular method described above, thus confirming Koch's postulates. Although A. rolfsii has been reported to cause disease on A. vera in India (Dubey and Pandey 2009), to the best of our knowledge, this is the first report of A. rolfsii causing southern blight on A. vera in China. Because A. rolfsii has a wide host range and is difficult to control (Punja 1985), occurrence of southern blight in China might be a serious threat for A. vera production and appropriate management strategies should be developed to control this disease.

First Report of Anthracnose Caused by Colletotrichum fructicola on Loropetalum chinense in China.

Loropetalum chinense Oliv. (Hamamelidaceae), an evergreen shrub or small tree, is widely used as a bonsai and landscape plant because of its aesthetic value. In addition, it has medicinal value, its leaves, flowers and roots can be used to treat diarrhea, coughs, hemorrhaging and burn (Zhang et al. 2013). In October 2020, leaf spot symptoms were observed on L. chinense distributed in Meiling Scenic Spot of Nanchang, Jiangxi Province, China (28.78°N, 115.83°E). We surveyed about 500 m2 of the mountain area which holds about 60 trees of L. chinense, with an incidence up to 20%. Initially, small gray-brown spots with dark brown edges appeared on the leaf surface, becoming large circular or irregular brown necrotic lesions over time. To isolate the pathogen, ten leaves of infected tissues were cut into 4 mm2 pieces, and surface disinfected with 75% ethanol for 30s and 1% hypochlorite for 1 min, rinsed three times with sterile water, plated on potato dextrose agar (PDA), and incubated at 25°C in the dark for 5 to 7 days. Five isolates with similar morphological characteristics were obtained. Conidia were hyaline, aseptate, smooth-walled, cylindrical with obtuse to slightly rounded ends, and their dimensions varied from 8.57 to 15.80 × 1.56 to 4.65 μm (n = 20). The morphological characteristics of the isolates matched the descriptions of Colletotrichum gloeosporioides species complex (Cai et al. 2009; Weir et al. 2012; Liu et al. 2015). For molecular identification, two representative isolates (JAUCC L003 and JAUCC L004) were selected for genomic DNA extraction, and the internal transcribed spacer (ITS), partial calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) were amplified, using primer pairs ITS1/ITS4, CL1C/CL2C, CHS-79F/CHS-345R, GDF1/GDR1, and ACT-512F/ACT-783R, respectively (White et al. 1990; Weir et al. 2012; Yang et al.2020). The sequenced loci (GenBank accession nos. ITS: MZ054401, MZ054400; CAL: MZ056750, MZ056754; CHS-1: MZ056751, MZ056755; GAPDH: MZ056752, MZ056756; ACT: MZ056753, MZ056757) exhibited up to 99% homology with corresponding sequences of C. fructicola Prihast., L. Cai & K.D. Hyde strains (GenBank accession nos. LC494271, MN525833, MN525854, MN525875 and MN525814). Concatenated sequences of the five genes were used to conduct a phylogenetic analysis using Maximum-Likelihood (ML) method in MEGA7. The isolates were identified as C. fructicola based on morphology and a multigene phylogenetic analyses. Pathogenicity of one representative isolate (JAUCC L003) was tested indoor by inoculating the top wounded leaves of six healthy L. chinense plants, rather than inoculating unwounded leaves due to the low incidence. Three leaves from each of three plants were punctured with flamed needles and sprayed with a conidial suspension (1 × 106 conidia/ml), and three leaves from each of other three plants were wounded and inoculated with mycelial plugs (5 × 5 mm3). Mock inoculations were used as controls with sterile water and PDA plugs on three leaves each. Treated plants were incubated in an artificial climate box at 25 °C, 90% relative humidity, with a 12-h photoperiod. All leaves inoculated with conidial suspension and mycelial plugs produced similar symptoms as described above 10 days postinoculation, whereas the mock inoculated plants remained asymptomatic. The fungus isolated from inoculated leaves was identical to the original pathogen on account of morphological and molecular data, confirming Koch's postulates, but not from the mock inoculated plants. Anthracnose disease caused by C. fructicola has been reported affecting numerous plants worldwide, including cotton, coffea, grape, citrus, mango, apple, pear, and cassava, among others (Guarnaccia et al. 2017, Oliveira et al. 2018). To our knowledge, this is the first report of C. fructicola causing anthracnose on L. chinense in China. This disease is significant concern in horticulture due to its impact on the aesthetics of ornamentals used in landscape plantings.

First Report of Colletotrichum truncatum Causing Anthracnose on Oxalis corniculata in China.

Oxalis corniculata L., which belongs to the family Oxalidaceae R. Br., is a very common perennial herb. It is usually planted on bare land or under the forest as landscaping plants, and the whole plant can be used for its medicinal values of clearing heat, detoxification and detumescence. In August 2019, typical symptoms of anthracnose on O. corniculata leaves were observed in the green belt on the campus of Shandong University of Technology (36.81°N, 117.99°E), Shandong Province, China. The disease incidence was above 40% by investigating more than 300 m2 of planting area. Most of O. corniculata are planted under the forest where the disease is found, mainly in the environment with high relative humidity and less ventilation. The infected leaves appeared initially as tawny oval or irregular spots, and then the lesions enlarged gradually until the leaves became dieback or wholly withered, which greatly reduced the landscape effect of O. corniculata. Diseased leaves were collected by cutting into small pieces and sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite (NaClO) for 60 s, rinsed with sterile deionized water for three times. Each air-dried tissue segment was cultured on potato dextrose agar (PDA) and incubated at 25℃ for 5 to 7 days in the dark (Zhu et al. 2013). Fifteen isolates were obtained from 20 symptomatic leaves and the cultures were initially gray white, subsequently became grayish to dark green after 7 days, with copious gray aerial mycelium and black microsclerotia. Three isolates were verified by the amplification of DNA sequences of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), histone H3 (H3) and chitin synthase (CHS1) genes, using the primer pairs GDF1/GDR1, ACT-512F/ACT-783R, CYLH3F/CYLH3R, and CHS-79F/CHS-234R (Damn et al. 2019, Fu et al. 2019, Liu et al. 2013), respectively. The sequenced genes (GenBank accession no. OK017473, OK159078, OK159076, OK159077) shared 99.62 to 100.00% nucleotide identity with the corresponding genes of Colletotrichum truncatum strain UASB-Cc-10 (GenBank accession no. KF322064.1, KF322055.1, KF322073.1, KF319059.1), respectively, which was consistent with the morphological identification (Sawant et al. 2012). Pathogenicity test was performed with six healthy O. corniculata plants infected with mycelial plugs (about 3 mm in diameter) of three C. truncatum isolates from a 5-day-old culture, while the negative controls on the same leaves were inoculated with sterile PDA plugs. All plants were placed in a greenhouse at 25 to 30℃ with 90% relative humidity. The experiment was conducted three times. Five days later, all inoculated leaves appeared brown sunken spots, whereas no symptoms appeared on negative controls. The same pathogens, C. truncatum, were identified from the inoculated leaves on the basis of morphological and molecular characteristics as described above, confirming Koch's postulates. To our knowledge, anthracnose caused by C. truncatum on O. corniculata is the first report in China. The discovery of this new disease is beneficial to the application and protection of O. corniculata, a popular landscape and medicinal plant.

First Report of Leaf Spot on Malania oleifera Caused by Botryosphaeria fabicerciana in China.

HomePlant DiseaseVol. 106, No. 6First Report of Leaf Spot on Malania oleifera Caused by Botryosphaeria fabicerciana in China PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Leaf Spot on Malania oleifera Caused by Botryosphaeria fabicerciana in ChinaYue Pan, Pengfei Wang, Wandong Chen, Guichai Yang, Yingjie Gu, Junwei Wang, and Juan WangYue Panhttps://orcid.org/0000-0002-2092-2366Eco-development Academy, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Pengfei WangCollege of Forestry, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Wandong ChenCollege of Forestry, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Guichai YangCollege of Garden and Horticulture, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Yingjie GuCollege of Forestry, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, Junwei WangSchool of Geography and Ecotourism, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this author, and Juan Wang†Corresponding author: J. Wang; E-mail Address: schima@163.comEco-development Academy, Southwest Forestry University, Kunming 650224, ChinaSearch for more papers by this authorAffiliationsAuthors and Affiliations Yue Pan1 Pengfei Wang2 Wandong Chen2 Guichai Yang3 Yingjie Gu2 Junwei Wang4 Juan Wang1 † 1Eco-development Academy, Southwest Forestry University, Kunming 650224, China 2College of Forestry, Southwest Forestry University, Kunming 650224, China 3College of Garden and Horticulture, Southwest Forestry University, Kunming 650224, China 4School of Geography and Ecotourism, Southwest Forestry University, Kunming 650224, China Published Online:11 May 2022https://doi.org/10.1094/PDIS-09-21-2038-PDNAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleMalania oleifera Chun & S. K. Lee is an endangered plant species in China with rich nervonic acid in seeds, and, thus, has important ecological and economic value (Fu 1992; Tang et al. 2013). Brown leaf spots were observed on 3-year-old M. oleifera plants in summer 2019 in Guangnan County, Yunnan Province, China (24°05′26″N, 105°05′07″E). A total of 100 trees within 0.3 ha of mountainous area were investigated, and eight (8%) showed disease symptoms. Initially, small, brown irregular spots appeared on the tip and margin of the leaf, and then gradually expanded to the whole leaf. Ten symptomatic leaves were randomly collected from different plants for fungal isolation. They were washed with running tap water, sterilized with 75% ethanol for 3 min and 1% NaOCl for 2 min, and rinsed in sterile distilled water three times. The leaves were cut into 5 × 5 mm2 segments and cultured on potato dextrose agar (PDA) at 25°C in the dark after air drying. In total, 12 morphologically similar fungal isolates were obtained. Colonies of the fungal culture initially appeared white, turned gray from the center between 3 to 6 days, and then became dark gray with cottony aerial mycelium 9 days later. To induce sporulation, the cultures were placed on 2% water agar (WA) with autoclaved pine needles and were incubated at 25°C under near-ultraviolet light as described by Chen et al. (2011). Globose conidiomata developed on pine needle surfaces after 10 days. Conidia were hyaline, unicellular, aseptate, fusiform with apex acute and base truncate, and 18.62 to 26.14 × 4.80 to 7.63 μm (average 21.77 × 5.91 μm, n = 50). The morphological characteristics resembled those of Botryosphaeria fabicerciana (S.F. Chen, D. Pavlic, M.J. Wingf. & X.D. Zhou) A.J.L. Phillips & A. Alves (Phillips et al. 2013). The internal transcribed spacer (ITS) region and the translation elongation factor 1-α gene (EF1-α) regions of a representative isolate (CGMCC3.20320) were amplified using primers ITS1F/ITS4 and EF1-728F/EF1-986R (Carbone and Kohn 1999; White et al. 1990), respectively. The obtained sequences (GenBank accession nos. MW642163 and MW651965) showed 99.80% (identity = 505/506) and 99.27% (identity = 272/274) similarity with the corresponding genes of ex-type CMW 27094 of Fusicoccum fabicercianum S.F. Chen, D. Pavlic, M.J. Wingf. & X.D. Zhou, now B. fabicerciana (GenBank accession nos. HQ332197 and HQ332213). The pathogenicity of the selected isolate (CGMCC3.20320) was evaluated by leaf inoculation of 1-year-old M. oleifera seedlings. Mycelial plugs (5 mm) of B. fabicerciana from a 7-day-old culture were applied on leaves wounded with sterile scalpels. Controls were inoculated with sterile PDA plugs. Each treatment was repeated six times. Inoculated and control plants were kept in a greenhouse at 25°C and 80% relative humidity, with a 12-h photoperiod. After 10 days, irregular brown spots appeared on all inoculated leaves, whereas there were no spots from control plants. Botryosphaeria fabicerciana was reisolated only from symptomatic leaves, fulfilling Koch’s postulates. Previous studies indicated that this fungus can cause stem lesions on Eucalyptus and trunk cankers on Carya cathayensis in China (Chen et al. 2011; Zhuang et al. 2021). To our knowledge, this represents the first report of B. fabicerciana causing leaf spot on M. oleifera in China.The author(s) declare no conflict of interest.

First Report of Anthracnose on Cinnamomun burmannii Caused by Colletotrichum scovillei in China.

Mei Pian tree belongs to a new physiological type of Cinnamomun burmannii discovered in the eastern part of the Guangdong province in China in 1987 (Chen et al. 2011). Although the external morphology of Mei Pian tree is similar to Cinnamomun burmannii, the leaves of Mei Pian tree, known as an important traditional Chinese medicine, are rich in natural D-borneol, which protects the heart, brain, and other organs, regulates the central nervous system, and promotes the absorption of other drugs (Yang et al. 2020; Fu et al. 2020). In April 2020, we found that the yield and quality of Mei Pian tree leaves were seriously threatened by anthracnose. Approximately, 40 - 60% of trees were infected in Pingyuan County, Meizhou City, Guangdong Province (N24°28'31.13", E115°50'50.02"). Small circular black spots were initially observed on infected leaves, and spots continued to grow and developed chlorotic margins and concentric rings with sunken areas. As the disease progressed, multiple spots were observed on almost all leaves. Four symptomatic leaves were collected and used for pathogen isolation. The areas of symptomatic and healthy-appearing leaf tissues at the margin of spots were surface-sterilized with 0.5% NaClO for 2 minutes and 70% alcohol for 30 seconds. The sterilized leaves were washed three times with sterile water, air dried, plated on potato dextrose agar (PDA) medium, and incubated at 28°C for 4 days in the dark. A total of six single-spored isolates were obtained and named from MPS-1 to MPS-6, respectively. Among those isolates, MPS-2, MPS-5, and MPS-6 were identical when cultured on PDA plate. The colonies were white to pale gray with dense aerial mycelia, and the reverse side of the colonies was light reddish brown. Conidia were cylindrical and measured 9.0 to 14.0 μm in length and 3.0 to 4.5 μm in width (n = 35). For molecular identification, the primers ITS1/ITS4, GDF/GDR , CHS-79F/CHS-345R, ACT-512F/ACT-783R and T1/Bt2b were used to amplify the partial regions of rDNA-ITS, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase(CHS1), actin (ACT) and β-tubulin (TUB2), respectively, from the genomic DNA extracted from fresh mycelia of MPS-2 (Damm et al. 2012). The resulting sequences were deposited in GenBank with accession numbers of MW091490, MW125584, MW125585, MW125586 and MW125587, respectively. The phylogenetic tree was generated by the maximum likelihood method of the MEGA 7 software using a concatenated alignment of ITS, GADPH, CHS1, ACT and TUB2 sequences. According to both morphological and sequence analyses, MPS-2 was identified as Colletotrichum scovillei (Damm et al. 2012, 2020). Pathogenicity tests were performed by inoculating healthy Mei Pian tree leaves with 5 mm PDA plugs containing actively growing mycelium of MPS-2 and wound-inoculated by spraying MPS-2 conidial suspension (106 conidia ml-1). Controls were inoculated only with sterile PDA plugs and ddH2O. All inoculated plants were maintained in a moist chamber (RH greater than 90%) at 25 °C, with an 8-h photoperiod under T5 LED lights. All inoculated leaves developed symptoms similar to those on naturally infected leaves after 5 days, but leaves on control plants remained asymptomatic. The fungus on the inoculated plants was identical in morphology to that found on the original sample collected in the field, thus fulfilling Koch's postulates. In previous studies, Colletotrichum scovillei also caused anthracnose on banana (Musa spp. AAA group), pepper (Capsicum annuum), and mango (Mangifera indica L.) in China (Zhou et al. 2016; Zhao et al. 2016; Qin et al. 2019). To our knowledge, this is the first report of Colletotrichum scovillei causing anthracnose on Cinnamomun burmannii in China and worldwide. The identification of C. scovillei as the causal agent of the observed anthracnose on C. burmannii is critical to the prevention and control of this disease in the future.

Identification of Leaf Spot and Stem Rot on Plectranthus verticillatus Caused by Rhizoctonia solani AG-4 HGI in China.

Plectranthus verticillatus (Swedish ivy) is a plant in the family Lamiaceae (Labiatae), native to South-east Africa, that is commonly grown as a houseplant in China. In June 2018, the initial symptoms water-soaked spots were found on the stem base of sixteen P. verticillatus plants(1-year-old plants) in a commercial greenhouse, situating at Daxing district, Beijing, China (116°22'E, 39°42'N), where there were 150 potted P. verticillatus plants obtained by cuttage propagation and grown in conventional nutrient soils. Almost amonthlater, the disease was more severe and leaves appeared symptoms. Infected leaves displayed water-soaked, darkbrown lesions and fell off easily. Symptoms on the stems were dark brown, rotted and lodging. In the survey, the incidence of affected plants was 70%. To isolate the causal agent, the infected leaf and stem tissues were cut into 3 mm segments and surface disinfected in 1% NaClO for 1 min, then rinsed three times with sterile distilled water and cultured on potato dextrose agar (PDA) at 25±1 °C with a 12-h photoperiod. After 2 days, hyphal tips were transferred to new PDA plates to obtain pure cultures. Six morphologically similar isolates (CC1 to CC6) were obtained from the symptomatic samples. All the fungal colonies were white initially, turned brown gradually, and formed irregular shaped sclerotia after 9 days of incubation on PDA. Observationofmicrostructures indicated that all the isolates resembled Rhizoctonia sp.(Sneh et al. 1991), including septate hyphal with right-angled branching, a slight constriction at the base of hyphal branches and 3 to 8 nuclei per cell. Total genomic DNA was extracted from pure isolates using a Fungal DNA Kit (GBCBIO, Guangzhou, China) and the internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1/ITS4 and sequenced (White et al. 1990 and Garibaldi et al.2019).The results showed that sequences of 6 isolates were identical and had a 100% identity with those of R. solani AG-4 HGI (GenBank accession no. MH172679 and MH172664). The phylogenetic analysis performed by the neighbour-joining method (MEGA 7 software) showed that the representative isolate (accession no. MZ723940) was assigned to group ofR. solaniAG-4 HG-I. The pathogenicity of these six isolates to the leaves and stems of P. verticillatus were conducted. Healthy P. verticillatus plants(1-year-old) were grown in 2-liter pots with sterile substrate in a greenhouse at 14-19°C (night)/23-28°C (day). After 7 days, five P. verticillatus plantsfor each isolate were inoculated with a 6-mm-diameter PDA plug taken from a 10-day-old PDA culture, which was placed onto leaf blade and the base of the stem. Five control pots were inoculated with sterile PDA plugs. All the inoculated plants were incubated in a glass cabinet under 90% humidity and 25°C for 2 days and were then moved back to the greenhouse with natural daylight conditions. The test was repeated three times. Ten days later, all inoculated leaves and stems appeared dark brown and water-soaked lesions similar to those observed on naturally infected plants, whereas control plants remained asymptomatic. The fungus that was reisolated from diseased tissues had the same morphological characters as the original isolates and confirmed to beR. solaniAG-4 HG-I by molecular identification. R. solani AG-4 HGI has an extensive host range, which had previously been recorded to cause leaf, root and stem rot on Plectranthus sp. in Florida in 1984 (Alfieri Jr. et al. 1984). To our knowledge, this is the first report ofR. solani AG-4 HGI infecting P. verticillatus in China. Its confirmation is a significant step toward management recommendations for growers.

First Report of Diaporthe foeniculina Associated with Grapevine Trunk Diseases on Vitis vinifera in Cyprus.

In June 2017, three vineyards were surveyed in the regions of Droushia (30-year-old, cv Mavro), Ineia (50-year-old, cv Xynisteri), and Lemona (15-year-old, cv Carignan) at the province of Paphos, Cyprus, with dieback incidence of 22%, 32%, and 14%, respectively. More specifically, affected grapevines exhibited severe dieback symptoms in spur and cordon positions, related to perennial cankers and internal brown discoloration. Thirty symptomatic samples, were surface-sterilized (95% ethanol) and wood chips were plated on potato dextrose agar (PDA), amended with streptomycin (500 μg/ml) at 25 °C for 3-5 days. Based on colony morphology (white to creamy color, with sparse aerial mycelium) and conidia production, nine Diaporthe-like isolates were obtained. For species identification, the internal transcribed spacer (ITS) region and β-tubulin (BT) genes were amplified using the primer pairs ITS1/ITS4 and Bt2a/Bt2b, respectively (Úrbez-Torres et al. 2008). Sequences of the isolates P101b, P114c, and P289a revealed >99.8% homology to NCBI voucher specimens of Diaporthe foeniculina (Sacc.) Udayanga & Castl. (ITS: CBS111553, MH050434; ΒΤ: KY511368, KF778966), and were deposited in the GeneBank (ITS: MT735646, MT737289, MT737287; BT: MT903969, MT903970, MT903971). Thus, 8.3% of the collected isolates (3 of 36) were identified as D. foeniculina, while the rest Diaporthe-like isolates were identified as D. ampelina. D. foeniculina isolates were also transferred on 2% water agar with sterile pine needles under a 12h/12h near-ultraviolet, light/darkness regime, at 25 °C, to induce sporulation (Guarnaccia and Crous 2017). Two weeks later, microscopic observations revealed dark brown to black, globose to sub-globose, ostiolate pycnidia (n = 30) 291 to 897 μm (595 ± 173) x 192 to 655 μm (364 ± 113) containing hyaline, unbranched conidiophores, bearing alpha- and beta-conidia in the form of yellowish cirri. Alpha-conidia were aseptate, hyaline, ovate to ellipsoidal, ranging (n=100) from 5.6 to 9.9 μm (7.5 ± 0.8) x 1.9 to 3.3 μm (2.7 ± 0.3). Beta-conidia were abundant, aseptate, hyaline, filiform, slightly curved (n = 100) from 22.4 to 35.3 μm (28.1 ± 2.5) x 1.2 to 2.3 μm (1.6 ± 0.2) (Udayanga et al. 2014). Pathogenicity tests were conducted with isolates P101b and P289a under greenhouse conditions (24-32 ⁰C, 70% RH). Ten 1-year-old rooted canes cv Mavro were inoculated with 4 mm mycelium plugs from actively growing cultures into wounds made by drilling between two internodes at the middle of the trunk. The same number of cuttings were inoculated with sterile PDA plugs, sealed with Vaseline, and wrapped with parafilm, serving as controls. Seven months later, all inoculated cuttings developed brownish wood discolorations (average 39 ± 13 mm), similar to naturally infected plants. No symptoms were observed in the controls. Successful re-isolations were made only from the inoculated cuttings and confirmed by colony morphology. Previously, D. foeniculina (as D. neotheicola) has been reported as grapevine wood saprophyte (Úrbez-Torres et al. 2014). It has also been reported to cause shoot canker and dieback in numerous hosts, including almond, avocado, citrus, and sweet chestnut (Annesi et al. 2016; Guarnaccia and Crous 2017; Diogo et al. 2010; Mathioudakis et al. 2020). This is the first record of D. foeniculina associated with grapevine trunk diseases (GTDs) in Cyprus. However, its relative importance as the causal agent of GTDs remains to be further investigated.

First Report of Fruit Canker Caused by Nothophoma quercina on Chinese quince in South Korea.

The Chinese quince (Pseudocydonia sinensis (Thouin) CK Schneid.) is a tree that is commonly distributed in all regions of South Korea and other Asian countries. The ripened yellow fruit contains medically active compounds (Hamauzu et al. 2005). It has been consumed as tea and candies and used in traditional medicine for treating asthma, cough, influenza, harsh throat, and tuberculosis and for liver protection (Chun et al. 2012). In the Kyungpook National University campus (Daegu, South Korea), fruit canker on the Chinese quince was ubiquitously observed during May-August 2020. The average disease incidence was around 30%-40%, which caused significant yield loss. Initially, minute, brown-to-rust-colored, unbroken, circular, necrotic areas appear, and in the advanced stage of infection, the epidermis tears open and tube- or aecia-like white structures are formed. Successively, the affected areas become necrotic and gradually enlarge to reach 3-5 cm in diameter. To isolate the causative pathogen, symptomatic tissues obtained from diseased fruits were surface-sterilized for 1 min with 70% ethanol, rinsed in sterile distilled water, and plated onto potato dextrose agar (PDA). The inoculated plates were incubated for 7 days at 25°C. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 15 isolates were obtained across 20 fruit trees investigated. The colonies on the PDA plates reached a diameter of 60-70 mm after 7 days at 25°C, spreading with a regular margin, aerial mycelium covering the entire colony, compact, white to pale gray in color, and solitary and globose pycnidia were produced after ten days. Conidiogenous cells were phialidic, hyaline, simple, smooth, doliiform to ampulliform, 3-5 × 3-4 μm; conidia were subglobose to oval or obtuse, thin-walled, smooth, aseptate, minute guttules, brown, 5.5-8 × 4-7 μm. These morphologies corresponded to those of phoma-like species. Sequence data for the 28S nrDNA, the internal transcribed spacer, β-tubulin, and RNA polymerase II subunit (White et al. 1990, Liu et al. 1999, Aveskamp et al. 2009) were obtained randomly for one of the pure isolates (EAH 2), which resulted in the GenBank accession numbers MW325675, MW325676, MW330391, and MW330390, respectively. The RAxML analysis (Stamatakis 2014) was run on the CIPRES Science Gateway portal of the combined sequence data of the isolate EAH 2 and the reference sequences obtained from GenBank. Analyses for the combined datasets were conducted with RAxML-HPC2 on XSEDE v. 8.2.10 using a GTR+GAMMA substitution model with 1000 bootstrap iterations. Results demonstrated that the isolate EAH2 formed a strongly support clade with the type isolates of Nothophoma quercina (Syd.) Q. Chen & L. Cai (basionym: Ampelomyces quercinus), which has been found on Quercus sp. in Ukraine (Chen et al. 2015). The procedure for Koch's postulates was followed to confirm fungal pathogenicity using 3-day-old mycelial disks. A total of 15 same-aged healthy fruits were divided into three groups, and each group received a different treatment. Artificial wounds were created on one group of fruits using a sterile pin, and a 5-mm mycelial plug of the fungus was placed on the injured tissues. Mycelial plugs were also placed on the surfaces of the sets of unwounded fruits. The remaining fruits were maintained as control and inoculated with sterile PDA plugs. The test was repeated three times. The wounded fruits exhibited symptoms after 8-10 identical to those observed in the field. The control group remained asymptomatic, and the morphology of the fungus reisolated from the inoculated fruits was similar to that of N. quercina. The phylogeny, together with morphological identification and inoculation results, confirmed the identity of the fungus as N. quercina (Chen et al. 2015). A previous study had also reported shoot canker caused by N. quercina in the Chinese quince (Yun and Oh 2016). However, to our knowledge, this is the first report of fruit canker caused by N. quercina in the Chinese quince.

First Report of Brown Rot Caused by Monilinia polystroma on Apple in Italy.

Brown rot is a common apple disease in Italy, caused by Monilinia fructicola, M. laxa and M. fructigena (Martini et al. 2013). In September 2020, in a 'Jeromine' apple orchard under integrated pest management located in Scarnafigi (44°39'N, 7°33'E, north-western of Italy), fruits (8.6%) showing brown to blackish firm lesions (6.0 to 8.0 cm diameter) were observed. In some fruits, rots were covered by yellowish stromata. Two isolates (MPI1; MPI2) were obtained from two symptomatic apples and cultured on potato dextrose agar (PDA) for 7 days at 25°C in 12-h light/12-h dark regime. A white-to-greyish mycelium with slightly undulate margins and irregular, black stromata developed on PDA after 12 days incubation. Conidia, observed in branched monilioid chains, (Suppl. Fig. 1) were one-celled, globose, limoniform, hyaline, 38 to 58 μm (mean: 48) × 20 to 44 μm (mean: 33). Based on morphology, the isolates were tentatively identified as Monilinia polystroma (G.C.M. Leeuwen) Kohn. A polymerase chain reaction with primers ITS1 and ITS4 was performed on internal transcribed spacer (ITS) region 1 and 2 and 5.8S gene. The sequenced amplicons (435 bp - 445 bp; GenBank Accession No. MW600854; MW600855) showed 100% identity to the reference isolate of M. polystroma (HQ846944) and to other isolates from apples (AM937114; JX315717) and plum (GU067539). The ITS region of M. polystroma had five nucleotides to distinguish it from the closest species M. fructigena (Zhu et al. 2016; MH862738) (Suppl. Fig. 2). The pathogenicity of both isolates was tested on mature 'Jeromine' apples (10.1% total soluble solids). Three replicates of six apples per isolate were surface disinfected with 1% NaClO. A mycelial plug (5 mm) from colony grown on PDA was inserted using a cork borer into a hole (6 mm) in each fruit (Vasić et al. 2016). Apples inoculated with sterile PDA plugs were used as control. Fruits were placed at 22 ± 1 °C, 85% relative humidity and 12 h light/12 h dark regime. Lesion size was measured after 3, 6 and 9 days of incubation. All inoculated fruits developed typical brown rot symptoms 6 days after inoculation and yellowish stromata appeared on the surface; control fruit remained healthy (Suppl. Fig. 3). The virulence of both isolates was statistically similar (Suppl. Table 1). M. polystroma was reisolated from all inoculated fruits and confirmed by molecular methods. This is the first report of M. polystroma on apple in Italy. M. polystroma was previously reported on apple in Hungary (Petróczy et al. 2009), on apricot in Switzerland (Hilber-Bodmer et al. 2012), on peach and pear in Italy (Martini et al. 2014; 2015), on plum in China (Zhu et al. 2016), and on apple in Serbia (Vasić et al. 2018). The emergence of this pathogen for pome and stone fruit production in Europe stimulates to study its biology and epidemiology, and its fitness and management, as compared to the other endemic Monilinia species.