First report of Southern blight, caused by Athelia rolfsii (syn. Sclerotium rolfsii Sacc.), on Hellebores in North America

Lenten rose (Hellebores hybridus) is an herbaceous perennial grown in landscapes and valued for early spring flowers and high levels of deer resistance. An additional benefit as a landscape plant comes from the high level of disease resistance, with only three fungal pathogens reported in North America. In August of 2021, a Lenten rose plant within a mature landscape in Silver Spring, MD, USA, (lat 39.116629 long 77.043198) was found with a collapsed canopy and brown stems near the soil line. Small clusters of brown sclerotia-like objects were seen along the stem. Samples of the sclerotia and diseased tissue were dipped in 70 percent ethanol for 15 sec, transferred to 5 percent NaClO for 30 sec, immersed in sterile water for one minute, then plated onto Potato Dextrose Agar. Sclerotia-like objects germinated and white mycelia covered the plates within five days of germination. Hyphae emerged from diseased tissue within two days and also grew rapidly. Cultures from sclerotia-like objects and diseased tissue produced white sclerotia which melanized to brown spherical sclerotia ranging in size from two to four mm. Culture samples (1 cm square) were excised from the culture plates and transferred to the base of three two-year old potted hellebore plants. Control plants had blocks of PDA placed at the base of the plants. Plants were placed in plastic bags for two days to maintain humidity, then maintained at room temperature without plastic bags. Petioles turned brown and collapsed within seven days of inoculation. White, fan-like hyphae were present along with maturing sclerotia. Samples from surface sterilized tissue and sclerotia produced the same culture morphology as the originally isolated cultures. Non-inoculated plants remained healthy, and the pathogen was not isolated from non-inoculated plants. Individual DNA samples were prepared from original cultures and the re-isolated cultures. Molecular identification was performed by amplification of the internal rRNA transcribed spacer region (ITS1/4, White et al. 1990 ), the large subunit rRNA (LSU), and the elongation factor-1A (EF1a). Amplification products were cloned into TOPO-TA pcr4 vector and sequenced (Macrogen USA). Sequences were submitted to GenBank for IT1/4 (OK172559) and LSU (OK172560). Homology to ITS1/4 was found with Athelia rolfsii (MN622806), to LSU with Athelia rolfsii (MT225781) and for EF1a with Athelia rolfsii (MW322687). This is the first report of Athelia rolfsii on Hellebores in North America (Farr, D.F & Rossman, A.Y. Fungal Databases, U.S. National Fungus Collections, ARS, USDA. Retrieved September 10, 2021). This report is unique in that few pathogens are known to infect Hellebores(Taylor et al. 2011) and southern blight is not commonly isolated in landscape plantings at Maryland latitudes. 1. White et al. PCR Protocols: A Guide to Methods and Amplifications. Academic Press, San Diego, 1990 2. Taylor, R.K., Romberg, M.K. & Alexander, B.J.R. A bacterial disease of hellebore caused by Pseudomonas viridiflava in New Zealand. Australasian Plant Dis. Notes 6, 28–29, 2011.

Isolation and molecular identification of fungi that cause stem rot disease in Bali's local legumes

Efforts to improve food security in Indonesia, especially in Bali, need to be supported by improvements in cultivation techniques, including the management of pests and diseases. Legume crops are often attacked by stem rot diseases which can cause decreased production and economic losses. This disease is generally caused by the soil-borne pathogenic fungus Sclerotium rolfsii or Athelia rolfsii. The macroscopic and microscopic morphologies of the two species of the fungus Sclerotium rolfsii and Athelia rolfsii are the same and difficult to distinguish, so molecular identification is needed to determine the species. The research aims to isolate and molecularly identify fungi that cause stem rot disease in local legume plants in Bali. Research methods include isolation of pathogenic fungi from legumes showing symptoms of stem rot disease in Bali, pathogenicity test, identification of the highest levels of virulent isolates, DNA extraction, DNA amplification by PCR, and electrophoresis, ITS region sequencing, and computer analysis sequences DNA. The results of isolation pathogens that cause stem rot disease in Bali's local legume plants obtained six fungal isolates coded SKT, SKB1, SKB2 SKB3, SKL and SKN isolates. SKT isolates had the highest virulence rate after the pathogenicity test of peanut plants. Molecular identification results show that SKT isolate is Athelia rolfsii, because it's in a clade with Athelia rolfsii fungi sequences in GenBank with 100% Bootstrap support.

First Report of Athelia rolfsii Causing Southern Blight on Sarcandra glabra in China

Sarcandra glabra, belonging to the family Chloranthaceae, is a Chinese medicinal plant. The whole dry plant can be used as a medicine; it is rich in bioactive phytochemicals that possess anti-bacterial, anti-inflammatory, anti-oxidant, and anti-tumor properties (Xie et al. 2020). The current market price of S. glabra is around US$5/kg, and the annual demand is 3 500 000~4 000 000 kg in China (Pan et al. 2007). To meet consumer demand for safe and high-quality herbal products, the artificial cultivation of S. glabra has been vigorously promoted. In 2020, it was observed that a plant disease affected S. glabra growth in Hunan province. The disease symptoms included constriction at the base of the stem, with decay and a white mycelium covering. The plants finally died with a disease incidence ranging from 15% to 20%. Using our previously published methods (Yi et al. 2019), one fungal isolate was isolated from the cultured symptomatic stem tissue on potato dextrose agar (PDA) medium and was named as Kb. The isolate was subsequently transferred into 70% glycerol for preservation. The Kb colony varied in color from white to light yellow. The septate hyphae grew rapidly on PDA medium, at approximately 25 mm/day, at 28 °C. On the fifth day, rhizomorphs were formed at the edge and on the center of the PDA plate. On the sixth day, sclerotia developed into a rapeseed shape (d = 1.2~2.3 mm) with a smooth surface, and with white, yellow, or chestnut brown coloring. Morphologically, Kb was similar to Sclerotium rolfsii (Sun et al. 2020). Vigorously growing aerial hyphae were selected for molecular identification. The internal transcribed spacers (ITS) were amplified using the primer pairs ITS1/ITS4 (Glass et al. 1995). BLAST searches against Genbank indicated that Kb’s ITS sequence shared 97% similarity with that of Athelia rolfsii (MN696630.1). Based on morphological and molecular characteristics, Kb was identified as A. rolfsii. The sequence was deposited in GenBank (MW288292). Pathogenicity tests were carried out using the following procedures. Three healthy S. glabra seedlings were inoculated at the stem base with a PDA plug (5 mm in diameter) covered with 5-day-old fungal mycelium cultured at 28 °C, while the remaining three seedlings were inoculated with distilled water only, as the control. Plants were incubated in a greenhouse at 28 °C. At 7 days post inoculation, the inoculated sites infected with the putative pathogen displayed identical constrictions as previously observed in the field. In contrast, the controls remained symptomless. The pathogen was reisolated from these infected seedlings, and its culture showed the same morphological and molecular traits as the original isolates. No pathogens were isolated from the control plants. Pathogenicity tests were repeated three times. Koch’s postulates were fulfilled. Although S. rolfsii has been previously reported to cause Southern Blight on mung bean crops in China (Sun et al. 2020), this is the first report on A. rolfsii causing similar symptoms of Southern Blight on S. glabra in Hunan Province, China. Identification of the pathogens causing each disease is important for the development of effective disease management strategies and for extensive artificial cultivation.

First Report of Root Rot on kiwifruits Caused by Athelia rolfsii in Hefei, China

Kiwifruits (Actinidia ssp.), known as “King of vitamin C”, have been wildly cultivated. In August 2020, about 15% of A. deliciosa (cv. Xuxiang) and A. macrosperma (rootstock) plants displayed symptoms typical of root rot at a farm in Hefei (117°25′E, 31°86′N), Anhui Province of China (Fig.1 a-b). Symptoms first appeared at the root and stem junction which were covered by cottony white mycelium during warm and humid summer. Then, the infected tissues were rotted, and subsequently the whole plant withered. Tan to brown sclerotia were observed on the basal stem epidermis and soil surface surrounding the stem (Fig.1 c-d). Infected plant tissues and sclerotia were collected for isolating the fungal pathogen. The samples were surface sterilized in 70% alcohol for 30 s, followed by 2% sodium hypochlorite for 3 min, washed five times with sterile double-distilled water (ddH2O), dried, placed on potato dextrose agar, and incubated at 25 °C in the dark. In total, twelve fungal isolates were obtained. The mycelia of all the isolates were white with a fluffy appearance (Fig.1 e). Sclerotia formed after 7 days were initially white (Fig.1 f) and gradually turned to dark brown (Fig.1 g) measuring 0.67 to 2.03 mm in diameter (mean = 1.367 ± 0.16 mm; n = 30). Hyphae were hyaline, septate. Some cells possessed multiple nuclei (Fig.1 h) and clamp connections (Fig.1 i). No spores were observed. For species-level identification, ITS1/ITS4 and EF1-983F/EF1-2218R primers were used to amplify the internal transcribed spacer regions (ITS) and translation elongation factor-1 alpha regions (TEF-1α), respectively (White et al. 1990; Rehner & Buckley 2005). Based on ITS and TEF-1α sequence analyses, all 12 isolates were categorized into two groups, group one including isolates NC-1 and NC-6~10 and group two containing NC-2~5 and NC-11~12. The length of ITS sequences for NC-1 (MW311079) was 684bp and 99% to 100% similar to Athelia rolfsii (MN610007.1, MN258360.1). Similarly, ITS sequences for NC-2 (MW311080) were 99% to 100% similar to A. rolfsii (MH858139.1; MN872304.1). Also, TEF-1α sequences of NC-1 (MW322687) and NC-2 (MW322688) were 96% to 99% similar to sequences of A. rolfsii (MN702794.1, GU187681.1, MN702789.1). Based on morphology and phylogenetic analyses (Fig.1 j&k), the isolates NC-1 and NC-2 were identified as Athelia rolfsii (anamorph Sclerotium rolfsii) (Mordue. 1974; Punja. 1985). To fulfill Koch’s postulates, ten sclerotia of NC-1 were incorporated into the soil near stems of healthy Xuxiang plants (Fig.2 a). Similar treatments were also used for plants of A. macrosperma or A. arguta (Fig.2 g&m). Each control group had the same number of plants (n=3) for inoculating with ddH2O. The plants were kept in an incubator with a relative humidity of 80% and temperature of 28°C with 16/8 hours light/dark photoperiod. After twenty days, the pathogen-inoculated plants developed similar symptoms of root rot observed in the field (Fig.2 b-d, h-j, n-o). Similarly, four days after inoculation with sclerotia, leaves developed water-soaked lesions (Fig.2 e, k&p). No significant difference in pathogenicity was observed between NC-1 and NC-2. Non-inoculated control plants remained disease-free (Fig.2 f, l&q). The pathogenicity experiments were repeated three times. The pathogen was re-isolated from infected tissues and sclerotia, and isolates were confirmed as A. rolfsii by the ITS sequences. A. rolfsii has been reported to cause root rot in kiwifruit in the USA (Raabe. 1988). To our knowledge, this is the first report A. rolfsii causing root rot on kiwifruits in China.

Bioactive secondary metabolites produced by the emerging pathogen Diplodia olivarum

A new cleistanthane nor-diterpenoid, named olicleistanone (1), was isolated as a racemate from the culture filtrates of Diplodia olivarum, an emerging pathogen involved in the aetiology of branch canker and dieback of several plant species typical of the Mediterranean maquis in Sardinia, Italy. When the fungus was grown in vitro on Czapek medium, olicleistanone was isolated together with some already known phytotoxic diterpenoids identified as sphaeropsidins A, C, and G, and diplopimarane (2-5). Olicleistanone was characterized as 4-ethoxy-6a-methoxy-3,8,8-trimethyl-4,5,8,9,10,11-hexahydrodibenzo[de,g]chromen-7(6aH)-one. When D. olivarum was grown on mineral salt medium it produced (-)-mellein (6), sphaeropsidin A and small amounts of sphaeropsidin G and diplopimarane. Olicleistanone (1) exhibited strong activity against the insect Artemia salina L. (100% larval mortality) at 100 μg mL-1 but did not exhibit phytotoxic, antifungal or antioomycete activity. Among the metabolites isolated (1-6), sphaeropsidin A (2) was active in all bioassays performed exhibiting strong phytotoxicity on leaves of Phaseolus vulgaris L., Juglans regia L. and Quercus suber L. at 1 mg mL-1. Sphaeropsidin A (2) also completely inhibited mycelium growth of Athelia rolfsii, Diplodia corticola, Phytophthora cambivora and P. lacustris at 200 μg per plug, and was active in the Artemia salina assay. Also in this assay, diplopimarane (5) and sphaeropsidin G (4) were active (100% larval mortality). Diplopimarane also showed antifungal and antioomycete activities. Athelia rolfsii was the most sensitive species to diplopimarane. Sphaeropsidin C (3) and (-)-mellein (6) were inactive in all bioassays. These results expand knowledge on the metabolic profile of Botryosphaeriaceae, and embody the first characterization of the main secondary metabolites secreted by D. olivarum.

First Report of Southern Blight on Polygonatum sibiricum Caused by Sclerotium delphinii in China

Polygonatum sibiricum Delar. ex Redoute is a plant species used for medicine and food. On one hand, its rhizomes have potential medicinal values such as enhancing immunity, anti-aging, anti-tumor and antibacterial as well as the effects of improving memory and reducing blood lipid and sugar. On the other hand, the rhizomes can also be used as raw materials for drinks, preserves, and health products (Su et al. 2018). The annual demand of P. sibiricum is about 3500-4000 tons in China, and the market demands and the price continue to rise in recent years (Su et al. 2018). In August 2019, there was an outbreak of southern blight in the P. sibiricum planting fields (N30°04′06″, E115°39′47″) of Luotian County in Hubei province of China. Approximately 30% of plants were affected in many fields (333.33 ha). We observed that the surface of the infected rhizome and the surrounding soils were covered with white hyphae and sclerotia. The hyphae gradually extended downward to the rhizomes, causing rhizome rot and leaf yellowing and wilting. Mycelial fragments and sclerotia from ten symptomatic rhizomes were collected in the fields and incubated directly on potato dextrose agar (PDA containing 50 µg/ml kanamycin) at 27℃. The fungal colonies were transferred to PDA after two days of cultivation. The white colonies were formed with fluffy aerial mycelia, which grew radially with an average growth rate of 20.54±0.52 mm/d (n=10). The color of the sclerotia was milky white at first, and then gradually turned to beige and yellow-brown. After two-week-incubation, the sclerotia became dark brown. Most of the sclerotia were spherical or nearly spherical, with round-bulges on the surface. The number of mature sclerotia produced per plate ranged from 8-23 (n=10), and the size ranged from 2.5×3.0 mm to 7.5×13.0 mm (5.95 ± 2.34×7.51 ± 2.88 mm; n=50). In addition, clamp connections were observed under the microscope. For molecular identification, genomic DNA was extracted from isolate HJ-1 using the CTAB method (Mahadevakumar et al. 2018). The internal transcribed spacer (ITS) regions of rDNA were amplified with the primers ITS1/ITS4 (White et al. 1990). The resulting showed ITS sequence (Accession number: MW049362) was 99.66% homology with Sclerotium delphinii according to the GenBank database. In addition, the second largest subunit of RNA polymerase II gene (RBP2) and part of the elongation factor 1-alpha (EF1-α) gene were amplified by using the primers RPB26F/RPB2-7CR (Liu et al. 1999) and EF595F/EF1160R, respectively (Wendland and Kothe 1997). RPB2 gene sequence was deposited in GenBank (Accession number: MW415935), and was 99.53% similarity identity to Athelia rolfsii isolate MSB5-1. TEF-1α sequence was deposited in GenBank (Accession number: MW415934), and was 91.35% similarity to S. delphinii strain Sd_405. Because there are very few reference sequences of RPB2 genes from S. delphinii in GenBank to compare, we choose the ITS and TEF-1α gene sequences to construct the concatenated phylogenetic tree by the neighbor-joining method (Tamura et al. 2013). The results showed that HJ-1 was clustered with S. delphinii isolates selected from NCBI database. Based on morphological and molecular characteristics, the fungus was identified as S. delphinii Welch (teleomorph Athelia rolfsii (Curzi) C.C. Tu & Kimbr). Pathogenicity tests were performed on the healthy leaves, roots, stems and plants (n=3) of P. sibiricum. Each sample was inoculated with one sclerotia produced from a fifteen-day-old colony and there was on wound treatment. These inoculated and control samples (treated with sterile water) were incubated in a moist chamber (25 ± 2 °C, RH 85%) (Mahadevakumar et al. 2018). Typical disease symptoms were apparent on leaves, stems, rhizomes and plants at 4, 6, 5 and 15 days post inoculation, respectively. Fulfilling Koch’s postulates, the fungal pathogens were isolated and purified from the inoculated site and were reconfirmed as S. delphinii based on the morphological features. To the best of our knowledge, this is the first report of S. delphinii causing southern blight on P. sibiricum in China. S. delphinii has a wide host range worldwide and often causes crop yield reduction. This study will be helpful for the prevention and control of P. sibiricum southern blight in the future.