First Report of Paraphoma chrysanthemicola Causing Crown and Leaf Rot of Atractylodes lancea in China

Atractylodes lancea is an important traditional Chinese medicinal plant whose rhizome is used for treating complaints such as rheumatic diseases, digestive disorders, night blindness and influenza. Jiangsu Province is the optimal cultivation location for high-quality A. lancea rhizome. Since June 2019, symptoms of crown rot and leaf rot were observed in about 10-20% of the A. lancea in a plantation (31° 36' 1" N, 119° 6' 40" W) in Lishui, Jiangsu, China. Lesions occurred on the stem near the soil line and on the leaves (Fig. 1A). Disease incidence reached approximately 80-90% by September, 2021 (Fig. 1B) and resulted in severe loss of rhizome and seed yields. For pathogen isolation, ten samples of symptomatic stem segments and ten diseased leaves were collected, surface-sterilized using 5% NaClO solution, rinsed with sterile water, cut into 0.5-2 cm segments, and plated to potato dextrose agar (PDA), and then incubated at 30°C in darkness. Pure cultures of four isolates showing morphological characteristics of Paraphoma spp. were obtained, identified as a single P. chrysanthemicola strain, and named LSL3f2. Newly formed colonies initially consisted of white mycelia; the five-day-old colonies developed a layer of whitish grey mycelia with a grey underside. 20-day-old colonies had white mycelium along the margin and with a faint yellow inner circular part with irregular radial furrows, and the reverse side looking caramel and russet (Fig. 1C). Pycnidia were subglobose (diameter: 5 to 15 μm; Fig. 1D). Unicellular, bicellular or strings of globose or subglobose chlamydospores developed from hyphal cells (Fig. 1E and 1F). The internal transcribed spacer (ITS) region and large subulin-28S of LSL3f2 were cloned using primers ITS1/ITS4 and LR0R/LR7 (Aveskamp et al. 2010, Li et al. 2013), and deposited in GenBank (OK559658 and OK598973, respectively). BLASTn search and phylogenetic analysis showed the highest identity between LSL3f2 and P. chrysanthemicola sequences (Fig. 1G) and confirmed LSL3f2 as P. chrysanthemicola. Koch’s postulates were completed using one-month-old vegetatively propagated A. lancea plantlets growing on autoclaved vermiculite/peat mixture at 26°C with a light/dark cycle of 12/12 hours. Each plantlet was inoculated with 5 ml of conidial suspension in water (1 × 108 cfu/ml) by applying to soil close to the plantlet, with sterile water used as a mock control (n = 10). By 20 days post-inoculation, inoculated plantlets showed a range of disease symptoms consistent to those observed in infested fields (Fig. 1H). Pathogenicity was additionally confirmed using detached leaves inoculated with a colonized agar plug of LSL3f2 or an uninoculated control comparison (diameter = 5 mm) and incubated at 26℃ in the dark. Five to seven days post-inoculation, detached leaves showed leaf rot symptoms including lesions, yellowing and withering consistent with those in infested fields, while control leaves remained healthy (n = 10, Fig. 1I). The pathogen was reisolated from the diseased plantlets and detached leaves, in both cases demonstrating the micromorphological characteristics of LSL3f2. P. chrysanthemicola has been reported to cause leaf and crown rot on other plants such as Tanacetum cinerariifolium (Moslemi et al. 2018), and leaf spot on A. japonicain (Ge et al. 2016). However, this is the first report of P. chrysanthemicola causing crown and leaf rot on A. lancea in China.

Transcriptome analysis and functional validation identify a putative bZIP transcription factor, Fpkapc, that regulates development, stress responses, and virulence in Fusarium pseudograminearum

Fusarium pseudograminearum is a soil-borne, hemibiotrophic phytopathogenic fungus that causes Fusarium crown rot and Fusarium head blight in wheat. The basic leucine zipper proteins (bZIPs) are evolutionarily conserved transcription factors that play crucial roles in a range of growth and developmental processes and the responses to biotic and abiotic stresses. However, the roles of bZIP transcription factors remains unknown in F. pseudograminearum. In this study, a bZIP transcription factor Fpkapc was identified to localize to the nucleus in F. pseudograminearum. A mutant strain (Δfpkapc) was constructed to determine the role of Fpkapc in growth and pathogenicity of F. pseudograminearum. Transcriptomic analyses revealed that many genes involved in basic metabolism and oxidation-reduction processes were down-regulated, whereas many genes involved in metal iron binding were up-regulated in the Δfpkapc strain, compared with the wild type. Correspondingly, the mutant had severe growth defects and displayed abnormal hyphal tips. Conidiation in the Fpkapc mutant was reduced, with more conidia in smaller size and fewer septa than in the wild type. Also, relative to WT, the Δfpkapc strain showed greater replaced by increased tolerance to ion stress, but decreased tolerance to H2O2. The mutant caused smaller disease lesions on wheat and barley plants, but the significantly increased TRI genes expression, compared with the wild type. In summary, Fpkapc plays multiple roles in governing growth, development, stress responses, and virulence in F. pseudograminearum.

First Report of Phytophthora nicotianae Causing Dianthus chinensis root rot and foliage blight in China

Dianthus chinensis is a popular ornamental plant that is widely cultivated in China. From May 2020 to 2021, root rot and foliage blight were observed on approximately 50% groundcover plants at several landscape sites of Xuanwuhu Park and Nanjing Railway Station, China. Symptoms of wilting and chlorosis appeared in the initial stage, and severe infection caused the whole plant to die . To recover the causal pathogen, infected root and leaf samples were cut into 5×5 mm2 squares, surface-disinfected in 70% ethanol for 30 sec, placed onto 10% clarified V8 PARP agar at 25°C . After three days, Phytophthora-like hyphae were visibly emerged from both root and leaf tissues and growing into cV8A. Individual hyphal tips were transferred to new cV8A plates to obtain a total of 10 pure isolates. Colony morphology of all isolates on cV8A had slightly radiate to stellate patterns with cottony aerial mycelia. After four or five days all isolates had identical morphological traits including papillate and noncaducous sporangia on cV8, hyphal swellings, and intercalary and terminal chlamydospores. A representative isolate Pni-dc7 was examined for morphological measurements. Sporangia were mostly ovoid and sometimes obpyriform, averaging 28.9±5.6 µm in length and 24.9±5.8 µm in width (n=30). Chlamydospores were abundant and spherical with an average diameter at 29.2 ± 0.3 µm (n=30). Oogonia were not observed. For sequence analysis, the internal transcribed spacer (ITS) regions and large subunit (LSU) of the nuclear ribosomal RNA gene complex were amplified using the primer pairs ITS1/ITS4 and NL1/NL4 , respectively, while the mitochondrial cytochrome c oxidase subunit II (coxII) gene was amplified using FM58/FM66 (Martin et al. 2003). The ITS sequence of isolate Pni-dc7 (GenBank Acc. No. MZ519893) had a 100% identity to those of P. nicotianae (MH219914, KU172524, MT065839). The LSU sequence (MZ573547) had a 100% identity to those of P. nicotianae (KX250514, MZ348950, HQ665198).The cox2 sequence (MZ519893) had a 100% identity to those of P. nicotianae (MH221078, KJ506439, JF707072). Based on morphological and molecular evidence, Pni-dc7 was identified as P. nicotianae. Pathogenicity tests were conducted using both detached leaves and whole plants. Asymptomatic leaves were collected from healthy plants.A 5×5 mm2 Pni-dc7-colonized cV8A plug was placed on each wound of five leaves. Sterile agar plugs were used for a non-inoculated control leaf. All six leaves were placed on a wet filter paper in a closed container at 25°C. All inoculated leaves had necrotic tissues around the wounds, the symptoms progressed from spots to the entire leaves after two days . The control leaves remained asymptomatic. In the whole-plant assay, a D. chinensis plant (approx. 0.3 m in height) was inoculated with 5 mL of zoospore suspension that was mixed into the potting soil(500g). Three plants were inoculated and control plants were treated with sterile distilled water. After two weeks all three inoculated plants in three repeats of the assay had root and crown rot and foliage blight, whereas all control plants remained asymptomatic. P. nicotianae was reisolated from all inoculated plants. This is the first report of P. nicotianae causing root rot and foliage blight on D. chinensis in China. Considering the importance of D. chinensis to both ornamental nursery and landscaping industries in China, diseased plants at the landscape sites were removed to prevent the spread of P. nicotianae to production sites and other landscape locations.

A megabirnavirus alleviates the pathogenicity of Fusarium pseudograminearum to wheat

Fusarium pseudograminearum is a phytopathogen that causes wheat crown rot disease worldwide. Fusarium pseudograminearum megabirnavirus 1 (FpgMBV1) was isolated from the hypovirulent strain FC136-2A of F. pseudograminearum as a novel dsRNA mycovirus belonging to the family Megabirnaviridae. Here we examined the effects of FpgMBV1 on colony morphology and pathogenicity of F. pseudograminearum. Through hyphal tip culture, we obtained virus-free progeny of strain FC136-2A, referred to as FC136-2A-V-.FpgMBV1 was transferred horizontally to another virus-free strain, WZ-8A-HygR-V-. The progeny that obtained through horizontal transfer was referred to as WZ-8A-HygR-V+. Colony morphology was similar between the FpgMBV1-positive and -negative strains. The ability to penetrate cellophane in vitro was lost and pathogenicity on wheat plants was reduced significantly in the FpgMBV1-positive strains relative to the FpgMBV1-negative strains. Microscopic observations showed a 6-h delay in the formation of appressoria-like structures in FC136-2A relative to FC136-2A-V-. And mycelium extension was significantly longer in wheat coleoptiles infected by WZ-8A-HygR-V- than in that infected by WZ-8A-HygR-V+ at 12 and 20 hours after inoculation (HAI). In addition, expression of five genes that encode cell wall-degrading enzymes differed significantly between FpgMBV1-positive and -negative strains at 12 and 20 HAI during early infection of wheat cells by conidia. This study provides evidence for the hypovirulence effect of FpgMBV1 on F. pseudograminearum and suggests that the underlying mechanism involves unsuccessful early infection and perhaps cell wall degradation.

Field Detection of Rhizoctonia Root Rot in Sugar Beet by Near Infrared Spectrometry

Rhizoctonia root and crown rot (RRCR) is an important disease in sugar beet production areas, whose assessment and control are still challenging. Therefore, breeding for resistance is the most practical way to manage it. Although the use of spectroscopy methods has proven to be a useful tool to detect soil-borne pathogens through leaves reflectance, no study has been carried out so far applying near-infrared spectroscopy (NIRS) directly in the beets. We aimed to use NIRS on sugar beet root pulp to detect and quantify RRCR in the field, in parallel to the harvest process. For the construction of the calibration model, mainly beets from the field with natural RRCR infestation were used. To enrich the model, artificially inoculated beets were added. The model was developed based on Partial Least Squares Regression. The optimized model reached a Pearson correlation coefficient (R) of 0.972 and a Ratio of Prediction to Deviation (RPD) of 4.131. The prediction of the independent validation set showed a high correlation coefficient (R = 0.963) and a root mean square error of prediction (RMSEP) of 0.494. These results indicate that NIRS could be a helpful tool in the assessment of Rhizoctonia disease in the field.

Identity and pathogenicity of fungi associated with root, crown and vascular symptoms related to winter squash yield decline

Winter squash (Cucurbita maxima cv. ‘Golden Delicious’) produced in Oregon’s Willamette Valley for edible seed production has experienced significant yield losses due to a soilborne disease. The symptoms associated with this disease problem include root rot, crown rot and vascular discoloration in the stems leading to a severe late season wilt and plant collapse. Through field surveys, Fusarium oxysporum, F. solani, F. culmorum-like fungi, Plectosphaerella cucumerina, and Setophoma terrestris were identified to be associated with diseased tissues, and each produced symptoms of root rot, crown rot or stem discoloration in preliminary pathogenicity trials. In this study, 219 isolates of these species were characterized by molecular identity analyses using BLAST of the ITS and EF1α genomic regions and by pathogenicity testing in outdoor, large-container trials. Molecular identity analyses confirmed the identity of isolates at 99 to 100% similarity to reference isolates in the database. In pathogenicity experiments, F. solani produced the most severe symptoms, followed by F. culmorum-like fungi, F. oxysporum, P. cucumerina, and S. terrestris. Some treatments of mixed species inoculum produced symptoms above what was expected from individual species. In particular, the mixture of F. culmorum-like fungi, F. oxysporum, and P. cucumerina and the mixture of F. culmorum-like fungi, F. solani, and S. terrestris had equally severe symptom ratings than that of F. solani by itself. Results indicate that this soilborne disease is primarily caused by Fusarium solani, but interactions among the complex of F. solani, F. culmorum-like fungi, F. oxysporum, and P. cucumerina, can exacerbate disease severity.

Phytophthora Root and Collar Rot of Paulownia, a New Disease for Europe

Paulownia species are fast growing trees native to China, which are being grown in managed plantings in several European countries for the production of wood and biomasses. In 2018, wilting, stunting, leaf yellowing, and collapse, as a consequence of root and crown rot, were observed in around 40% of trees of a 2-year-old planting of Paulownia elongata × P. fortunei in Calabria (Southern Italy). Two species of Phytophthora were consistently recovered from roots, basal stem bark, and rhizosphere soil of symptomatic trees and were identified as Ph. nicotianae and Ph. palmivora on the basis of both morphological characteristics and phylogenetic analysis of rDNA ITS sequences. Koch’s postulates were fulfilled by reproducing the symptoms on potted paulownia saplings transplanted into infested soil or stem-inoculated by wounding. Both Phytophthora species were pathogenic and caused root rot and stem cankers. Even though P. palmivora was the only species recovered from roots of naturally infected plants, in pathogenicity tests through infested soil P. nicotianae was more virulent. This is the first report of Phytophthora root and crown rot of a Paulownia species in Europe. Strategies to prevent this emerging disease include the use of healthy nursery plants, choice of well-drained soils for new plantations, and proper irrigation management.