A new species, Chloridium paucisetosum, was isolated from soil in Guizhou Province, China. The species is distinguished from other Chloridium species by its cylindrical or lageniform conidiogenous cells producing obovoid or ellipsoidal conidia without mucoid appendages, and apical branched conidiophores. Phylogenetic analysis based on combined ITS and LSU sequence data showed that C. paucisetosum formed a monophyletic clade and clearly separated from other Chloridium species. It is compared with other similar species and their phylogenetic relationships are discussed. Morphology and phylogenetic analyses support the introduction of the new species C. paucisetosum. In addition, a previously described species Phialocephala humicola is synonymized under the genus Chloridium.

Background: Rapidly identifying candidate genes potentially involved in stress tolerance is crucial for functional genetic studies in non-model plant species. Agrobacterium-based transient assays can be conducted in many plants using fleshy tissues such as leaf/fruit/petal. However, such assays are not feasible in perennial ryegrass (Lolium perenne L.) and most other grass species since these plants have no fleshy tissue to be readily infected. Results: In this study, we developed a Protoplast-based Rapid stress regulatory gene Identification Assay (briefed as ‘PRIDA’) to rapidly identify genes in the regulation of plant heat and oxidative tolerance in perennial ryegrass. Using ryegrass mesophyll protoplasts, vectors harboring target genes or their RNAi hairpin structures driven under the maize ubiquitin promoter were transformed into protoplasts to transiently over-express or suppress the target genes. The transformed protoplasts were then subjected to optimized heat (35 to 37 ℃) or oxidative (50 to 75 mM H2O2) stress, and their viabilities were immediately recorded under a microscope after 0.1% Evans blue dye staining. The validity and reliability of PRIDA were verified by transiently knocking-down LpNOL/LpSGR and over-expressing LpTT3.1/LpTT3.2 that led to significantly compromised or enhanced protoplast viability after heat or H2O2-induced oxidative stresses. Finally, using ‘PRIDA’, we identified three heat-induced differentially expressed E3 ligase genes as potential regulators in plant heat and/or oxidative stress tolerance. Conclusion: This protoplast-based ‘PRIDA’ can be used as a versatile assay to rapidly identify and characterize candidate regulatory genes in heat and oxidative stress that will accelerate molecular genetic studies in perennial ryegrass. This assay can be adapted to other non-model plant species after adjusting abiotic stress parameters for further research and the development of improved cultivars.

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Faba bean (Vicia faba L.) is a cool-season legume crop, planted worldwide as an essential source of protein-rich foods, vegetables, and animal feeds. In China, the total cultivated area of faba bean in 2019 was 839,618 square hectometers (hm2) and the production was 1,740,945 tons (t) (Ji et al. 2022). In May 2021, a leaf spot disease on faba bean plants with about 80% disease incidence was observed in a 0.3 ha commercial field located at Zunyi City (27°31′43.80″ N, 106°23′34.27″ E), Guizhou Province, China. The leaves of the early affected plants appeared circular dark brown spots, which then rapidly develop into large irregular shaped lesions if conditions remain favorable. Severe infection can result in extensive defoliation of plants and lesions on pods. Symptomatic leaves were collected and cut into small pieces, surface sterilized with 75% ethanol for 30 s followed by 2% NaClO for 1 min, rinsed with sterile distilled water three times, and incubated on PDA plates amended with streptomycin sulfate (0.5 mg/L) at 25°C for 2–4 days. Two purified cultures were obtained through single-spore culture. Colonies on PDA attaining 62 mm diam after 2 weeks, white or pale red, edge undulate, with dense aerial mycelium on the surface, fruiting bodies black to reddish brown. Conidia fusoid to ellipsoid, 4-septate, straight to slightly curved, 18.5–22 × 6–7 μm (av. = 20 × 7 μm, n = 30); basal cell obconic, hyaline, 3.5–5 μm long, with a single appendage, 2.5–6 μm; three median cells doliiform, verruculose, olivaceous with slightly red (second cell from base 4–5 μm long; third cell 4.5–5.5 μm long; fourth cell 3–5.5 μm long); apical cell conical, hyaline, 2.5–4 μm long, with 1–3 tubular appendages, 13–22.5 μm long. The morphological characters of our studied specimens fit well with Pestalotiopsis rosea (Maharachchikumbura et al. 2012). For molecular identification, the internal transcribed spacer (ITS) region, partial β-tubulin (tub2) and translation elongation factor 1-alpha (tef1-α) genes were amplified and sequenced using primer pairs ITS5/ITS4 (White et al. 1990), T1/Bt-2b (Glass and Donaldson 1995) and EF1-728F/EF-2 (Carbone and Kohn 1999). The DNA sequences of two isolates GUCC 195257 (OP364052, OP391714, OP391713) and GUCC 195258 (OP364053, OP391716, OP391715) were deposited in GenBank. The BLAST searches revealed that these sequences had 99% (537/539 bp), 100% (453/453 bp), 99% (591/593 bp), 99% (537/539 bp), 100% (453/453 bp) and 99% (574/576 bp) nucleotide identity to the ex-type strain of P. rosea (JX399005, JX399036, JX399069), respectively. In addition, multi-locus phylogenetic analysis showed that both isolates clustered with P. rosea with full statistical support. The phylogenetic relationship of Pestalotiopsis species supported the identification of our isolates as P. rosea. In the pathogenicity test, the leaves of ten healthy 2-week-old faba bean plants were spray inoculated with a conidial suspension (1 × 106 conidia/ml) of the two isolates. Another set of five plants that were sprayed with sterilized distilled water served as the controls. Treated plants were kept at 25°C in a greenhouse with a photoperiod of 12 h and 70% relative humidity. After one week, all inoculated leaves showed symptoms similar to those of the infected faba bean observed in the field, whereas controls were symptomless. The pathogenicity test was performed twice with similar results. The fungus was reisolated from the inoculated plants and identified as P. rosea by morphological and molecular evidence, thus confirming Koch’s postulates. To our knowledge, this is the first report of P. rosea causing leaf spot on faba bean in the world. Pestalotiopsis species are well-known phytopathogens that can cause a variety of diseases, including leaf spots, chlorosis, and various postharvest (Wang et al. 2019). The results of this study not only contribute to accurately identify this disease in the fields of faba bean production, but also provide an important reference for developing specific control measures.

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