Distribution and diversity of myxomycetes in Tiantangzhai National Forest Park, China

Although myxomycetes are ubiquitous in terrestrial ecosystems, studies on their distribution and diversity in subtropical humid forests are still lacking. Field collections and moist chamber cultures were conducted from May to October within a two-year period in the Tiantangzhai National Forest Park of China. A total of 1,492 records representing 73 species belonging to 26 genera were obtained, of which 243 records/37 species were from field collections, and 1,249 records/52 species were from moist chamber cultures. Among the specimens obtained by culturing, 896 records/38 species and 353 records/37 species were obtained from living bark and ground litter, respectively. ANOVA showed that the sampling months had significant impacts on collection of myxomycetes from field and those that inhabit litter. An LEfSe analysis indicated that Arcyria was significantly abundant in August, while Stemonitis and Physarum were more abundant in July when collected from field. An RDA analysis showed that temperature was the main factor that affected the litter-inhabiting myxomycetes. The ANOVA indicated that forest type was the significant factor for bark-inhabiting myxomycetes. Diderma effusum was primarily obtained from mixed forests, while Clastoderma debaryanum and Colloderma oculatum were more common in coniferous forests. The RDA analysis indicated that the vegetation, pH, water retention, and elevation were the primary factors that affected the bark-inhabiting myxomycetes.

First report of Curvularia lunata causing Curvularia leaf spot of corn in Delaware

In mid-August 2020, small circular tan-colored lesions were detected in leaves of corn, Zea mays, in Sussex county Delaware. Symptoms were observed in many fields, affecting multiple hybrids that were at approximately the dough growth stage (R4). Lesions were present on most plants and individual ear leaf severity ratings ranged from 3 to 14 %. Symptomatic corn leaves were selected from the field and kept in a moist chamber for 24 hrs at 25 °C. In lesions, melanized spores were observed ranging from 21.94 µm to 30.51 µm in length and 7.83µm to 11.70µm in width (n=20). One cm2 leaf sections were extracted and sterilized in a 0.85% sodium hypochlorite solution for 30s followed by a sterile water rinse for 30s. Leaf pieces were then plated onto potato dextrose agar amended with 50µg/ml penicillin-G-sodium salt and streptomycin sulfate. Petri dishes were incubated at 25 °C with a 12-hr photoperiod for 14 days. Colonies were brown-black to lighter gray in color. Media was stained from orange, to dark brown, or not at all. Conidia were melanized and curved with three transverse septa matching the size above. Colony and spore morphology were consistent with the description of Curvularia lunata (Garcia-Aroca et al. 2018). Pure cultures were obtained, and a representative isolate was sequenced to confirm fungal identity by amplifying the internal transcribed spacer (ITS) region with primers ITS4 and ITS5. BLAST search results confirmed 100% similarity (483/531 bp) to the C. lunata reference sequence (MK623264). The sequence was deposited in GenBank as accession number MW794323. To complete Koch’s postulates, corn (hybrid: Hubner H6187RCSS) was planted into 10 cm pots and maintained at 25°C and a 12 hr photoperiod. A conidial suspension was made by soaking a Petri dish of a 3-week-old fungal colony with 20 ml of sterile water and a drop of Tween-20 solution then, scraping mycelia to dislodge spores. The conidial concentration was calibrated to 5 x 105 spores/ml and 15 ml were applied to each whorl of four three-collar stage plants using a Preval sprayer (Nakoma Products, Bridgeview, IL). Four plants were inoculated with 15 ml sterile DI water as a control. This experiment was repeated twice. Each plant was moved to an incubator and covered with a plastic bag for 24 hr to maintain humidity. Conditions in the incubator were maintained at 25 °C with a 12-hr photoperiod. Inoculated plants displayed small, oval-shaped lesions within four days. No symptoms were observed on the control plants. Symptomatic leaves were harvested and placed into a moist chamber for 24 hrs to sporulate and lesions were plated onto PDA as described above. Culture morphology was consistent with the original isolate, with spores slightly larger ranging from 22.35 µm to 39.29 µm in length, and 8.36 µm to 11.69 µm in width (n=20). Isolates obtained from inoculated plants were sequenced and maintained 100% identity with the reference sequence described above. C. lunata was first reported in Louisiana in 2017 (Garcia-Aroca et al. 2018) and in Kentucky in 2018 (Anderson et al. 2019). This is the first report of Curvularia leaf spot on corn in Delaware. Symptoms developed late in the season, so it is unlikely that yield was affected in 2020. However, the economic impact of this disease in the United States is still unclear, it will be important to monitor potential impacts of this disease in Delaware corn production. References 1.Anderson NR, et al., 2019, Plant Disease, 103, 2692, doi: 0.1094/PDIS-03-19-0629-PDN. 2. Garcia-Aroca T, et al., 2018, Plant health progress, 19, 140, doi: 10.1094/PHP-02-18-0008-BR.

Five new Myxomycetes (Myxogastria) records from Turkey

Myxomycetes samples were obtained from Adana and Hatay provinces of Turkey. As a result of field and laboratory studies five myxomycetes species are reported as new records. Three of the new records (Arcyria cerradensis, Craterium aureonucleatum and Diderma spumarioides) were found as natural sporophore in the area. The other two species (Cribraria spinispora and Perichaena quadrata) were grown in the laboratory by moist chamber culture method. Short descriptions, habitats, localities, collection dates, fungarium numbers and images of the taxa are given. This work has contributed to Myxobiota of Turkey.

Diderma aglomerospora (Didymiaceae, Physarales, Myxomycetes), a new species from Brazil

Diderma aglomerospora is here proposed as a new species based on morphological analyses. The new species resembles D. badhamioides by possessing clustered spores but differs from it by the absence of a columella and narrower filaments of the capillitium. The type material was developed in moist chamber assembled with bark of live trees from a restinga forest environment in the Guadalupe Environmental Protection Area, Pernambuco, Northeast Brazil. Sporocarps are described and illustrated, and comments on characteristics of the microhabitat are provided.

First Report of Fusarium wilt of Coleus forskohlii Caused by Fusarium oxysporum in China

Coleus forskohlii (Wild) Briq. is an aromatic plant in the Lamiaceae family cultivated primarily in India, Sri Lanka, Nepal and China (Yunnan Province). This herb is considered to have medicinal properties and the whole plant can be used to treat asthma, cancer and other diseases with remarkable efficacy. Due to the high medicinal and economic value of C. forskohlii, it has been introduced to Tongcheng (N29°18′12.24″, E113°53′59.36″), Hubei Province for cultivation. However, severe Fusarium wilt disease of C. forskohlii has been epidemic in Tongcheng since 2018 with a disease incidence of 5 to 30% in surveyed fields. This disease is characterized typically by root rot, vascular discoloration and leaf wilting of C. forskohlii (Fig 1), resulting in progressive plant death. Ten diseased plants were collected from the fields and the roots and stems were rinsed in 70% ethanol for 5 min and samples at the junction of disease and healthy tissues (0.5 × 0.5 cm2) were cutted and placed on potato dextrose agar (PDA) for fungal isolation in a dark chamber at 28°C. Eventually, ten pure isolates were obtained from hyphal-tip followed by single-spore purification on PDA. Seven of the purified isolates showed white aerial mycelium initially and secreted orange-brown pigment 8 days after incubation. Macroconidia were falciform, hyaline, three to five septate, ranging from 2.02 to 4.17 (mean 2.98 µm) × 10.05 to 21.90 µm (mean 12.04 µm) in size (n = 30) (Fig 2). These morphological characteristics resembled Fusarium oxysporum. (Leslie and Summerell 2006) and we selected one of them for molecular identification. Genome DNA was extracted from isolate (RS-4) using the CTAB method (Mahadevakumar et al. 2018). The translation elongation factor 1 alpha (EF-1α) DNA sequence was amplified using primers EF1/EF2 (Geiser et al. 2004), and the second largest subunit of RNA polymerase II (RPB2) DNA sequence was amplified using primers fRPB2-5F/fRPB2-7cR (Liu et al. 1999). The obtained EF-1α sequence of RS-4 (MW219142) showed 100% identity with that of F. oxysporum (FD_01376) (FUSARIUM-ID database). RPB2 sequences of RS-4 (MW219143) showed 100% identity with F. oxysporum (FD_01679) (FUSARIUM-ID database). Moreover, a phylogenetic tree of the EF-1α gene sequence of RS-4 was constructed based on the Neighbor-Joining method in MEGA7 software (Tamura et al. 2013) and revealed that strain RS-4 was closest to F. oxysporum (Fig 2). To test the pathogenicity of RS-4, six healthy leaves of C. forskohlii were collected and inoculated either with the colonized PDA discs (diameter, 5 mm) of RS-4 or control PDA discs, in a moist chamber at 25 ± 2°C. Five days later, brown-black lesions were observed on all inoculated leaves. However, the non-inoculated leaves were maintained asymptomatic. For in vivo pathogenicity test, twenty-day-old C. forskohlii plants (n=3) were inoculated with 106 spores/ml of the RS-4 at a position approximately 1 cm above the soil. Three seedlings treated with sterile water were used as controls. These inoculated and control seedlings were incubated in a moist chamber (25 ± 2 °C, RH 85%). Three days later, typical Fusarium rot symptoms were observed on all inoculated seedlings with rotten stems and withering leaves (Fig 2). Fungal pathogens were re-isolated from the inoculated sites of in vitro and in vivo inoculations by repeating the above isolating operation, and were reconfirmed through morphological features. This is the first report of F. oxysporum causing root rot on C. forskohlii in China. F. oxysporum is one of the most economically important fungal pathogens causing vascular wilt on a wide range of plants worldwide (Dean et al. 2012). The identification of F. oxysporum as the causal agent of the observed Fusarium wilt on C. forskohlii, is critical to the prevention and control of this disease in the future. Acknowledgement This research was supported by funding from the Key project at the central government level titled, “The ability to establish sustainable uses for valuable Chinese medicinale resources” (2060302) Reference Dean, R., et al. 2012. Mol. Plant. Pathol. 13: 414. https://doi.org/10.1111/j.1364-3703.2011.00783.x. Geiser, D. M., et al. 2004. Eur. J. Plant Pathol. 110: 473. https://doi.org/10.1023/B:EJPP.0000032386.75915.a0. Leslie, J. F. and Summerell, B. A. 2006. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, U.K. Liu, Y. J., et al. 1999. Mol. Biol. Evol. 16: 1799. https://doi.org/10.1093/oxfordjournals.molbev.a026092 Mahadevakumar, S. et al. 2018. Eur. J. Plant Pathol. 151:1081. https://doi.org/10.1007/s10658-017-1415-2. Tamura, K., et al. 2013. Mol. Biol. Evol. 30: 2725. https://doi.org/10.1093/molbev/msw054.

Myxomycetes biodiversity in Gaziantep Province (Turkey) with four new records

Myxomycetes were cultured in moist chambers using substrate material collected in Gaziantep province, Turkey, during 2017–2019. Fruit bodies of wild myxomycetes were collected at ten locations. Rotten or live tree bark, leaves, debris, vegetable, and animal material, which were considered likely to contain spores, were also collected. Natural samples were immediately dried, and potential spore-bearing material was kept in a warm and humid environment with the moist chamber technique. A total of 537 samples were studied and 203 myxomycetes isolates were obtained, 33 of which were natural samples, 76 were obtained with the moist chamber technique and 94 were obtained both naturally and with the moist chamber technique in the laboratory. Six orders, 9 families, 16 genera and 42 species were identified in 3 subclasses. All species were new in Gaziantep province and four myxomycetes were identified as new records in Turkey; Didymium atrichum Henney & Alexop., Didymium serpula Fr., Craterium obovatum Peck and Physarum bivalve Pers. were added to the Turkish mycota.

First data on Myxomycetes of the State Nature Preserve “Sut-Khol”, Republic of Tuva (Tyva), Russia

First records of Myxomycetes from the Republic of Tuva, Russia, are presented. We identified 24 species of myxomycetes from 12 genera, 7 families and 4 orders in the State Natural Customer “Sut-Khol”. For the Republic of Tuva, 14 species of myxomycetes have been identified for the first time. Arcyria globosa, first identified on the bark of a live Сaragana in the Asian part of Russia using the “moist chamber”. We have also identified a rare species of Echinostelium fragile on the bark of a live Pinus sylvestris.

Myxomycetes associated with a residential ecosystem

The myxomycetes associated with samples of the bark of living trees, ground litter, twigs, and aerial litter collected from a residential ecosystem were investigated with the use of moist chamber cultures. A total of 26 species representing 13 genera were recovered from 100 cultures prepared with samples of the four different types of substrates. The distribution patterns and ecology of these species are discussed in the context of the senior coauthor’s more than 40 years studying myxomycetes.