RESUMO
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°31'43.80â³ N, 106°23'34.27â³ 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 ß-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.
RESUMO
Rosa roxburghii Tratt. is widely applied in food, cosmetics, and traditional medicine, and has been demonstrated to possess diverse bioactivities. Plant endophytic fungi are important microbial resources with great potential for application in many fields. They not only establish mutualistic symbiosis with host plants but also produce a variety of bioactive compounds. Therefore, in the present study, endophytic fungi were isolated from R. roxburghii, the diversity and antimicrobial activities were evaluated. As a result, 242 strains of endophytic Sordariomycetes were successfully isolated. Multigene phylogenetic analyses showed that these isolates included eight orders, 19 families, 33 genera. The dominant genera were Diaporthe (31.4%), Fusarium (14.4%), Chaetomium (7.9%), Dactylonectria (7.0%), Graphium (4.5%), Colletotrichum (4.1%), and Clonostachys (4.1%). For different tissues of R. roxburghii, alpha diversity analysis revealed that the diversity of fungal communities decreased in the order of root, fruit, stem, flower, leaf, and seed, and Clonostachys and Dactylonectria exhibited obvious tissue specificity. Meanwhile, functional annotation of 33 genera indicated that some fungi have multitrophic lifestyles combining endophytic, pathogenic, and saprophytic behavior. Additionally, antimicrobial activities of endophytic Sordariomycetes against Lasiodiplodia theobromae, Botryosphaeria dothidea, Colletotrichum capsici, Pyricularia oryzae, Rhizoctonia solani, Fusarium oxysporum, Pseudomonas syringae, Pantoea agglomerans, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa were screened. Dual culture test assays showed that there were 40 different endophytic species with strong inhibition of at least one or moderate inhibition of two or more against the 12 tested strains. The results from the filter paper diffusion method suggested that extracellular metabolites may be more advantageous than intracellular metabolites in the development of antimicrobial agents. Eleven isolates with good activities were screened. In particular, Hypomontagnella monticulosa HGUP194009 and Nigrospora sphaerica HGUP191020 have shown promise in both broad-spectrum and intensity. Finally, some fungi that commonly cause disease have been observed to have beneficial biological activities as endophytic fungi. In conclusion, this study showed the species composition, alpha diversity, and lifestyle diversity of endophytic Sordariomycetes from R. roxburghii and demonstrated these isolates are potential sources for exploring antimicrobial agents.
RESUMO
BACKGROUND: Rosaroxburghii Tratt., a deciduous shrub of the family Rosaceae, is usually used as food and medicinal materials and also cultivated as an ornamental. Plant endophytic fungi are a large class of microbial resources not fully researched, with great potential applications. Two strains of Ascodesmis and Talaromyces were isolated during a survey of biodiversity on endophytic fungi of R.roxburghii in China. Multigene phylogenetic analyses showed that each of the two fungi formed a distinct lineage and separated from known congeneric species and they are proposed as two novel taxa. NEW INFORMATION: Ascodesmisrosicola sp. nov. usually has one or two conspicuous simple or branched ridges extending to the majority of the ascospore surface and remarkably small asci, distinguishing it from the previously-described species in the genus Ascodesmis. Talaromycesrosarhiza sp. nov., of the section Talaromyces, is closely related to T.francoae. It differs from the latter by having both monoverticillate and biverticillate conidiophores, while those of T.francoae are biverticillate. Both novel endophytes are illustrated and described.