In this research, the volatile organic compounds (VOCs) produced based on C. fimbriata exhibited strong antifungal activity against the fungal pathogen A. alternata . Our aim is to explore their bacteriostatic components.

Species of Ceratocystis and allied genera are pathogens of many trees, including Cunninghamia lanceolata. During a survey of 2020, we found a serious wilt disease of C. lanceolata in Yunnan Province, China. Three different fungi resembling Ceratocystis and allied genera were consistently isolated from discoloured foliage and stems on C. lanceolata. Morphological and DNA sequence comparisons based on 60S and LSU gene regions showed that the pathogens were Ceratocystis and related species. We included 4 isolates identified as C. acaciivora = C. manginecans, 10 isolates identified as Berkeleyomyces basicola ≡ Thielaviopsis basicola, and 1 isolate identified as Chalaropsis sp., from 3 geographical locations. Pathogenicity tests on potted plants showed that all three species were pathogenic. To our knowledge, this is the first report Ceratocystis acaciivora,B. basicola and Chalaropsis sp. causing C. lanceolata wilt in China.

Microorganism-produced volatile organic compounds (VOCs) are considered promising environmental-safety fumigants in food preservation. In this study, the VOCs from fungal Ceratocystis fimbriata strains (WSJK-1, Mby) were tested against postharvest fungi Monilinia laxa, Fusarium oxysporum, Monilinia fructicola, Botrytis cinerea, Alternaria solani, and Aspergillus flavus in vitro. The mycelial growth was significantly inhibited, in particular M. fructicola and B. cinerea (76.95, 76.00%), respectively. VOCs were identified by headspace solid-phase microextraction coupled with Gas Chromatography–Mass Spectrometry (HS-SPME-GC–MS); 40 compounds were identified. The antifungal activity of 21 compounds was tested by the minimum inhibitory concentrations (MIC) value. Benzaldehyde, 2-Phenylethanol, and 1-Octen-3-ol showed strong antifungal activity with the MIC in vitro ranging from 0.094 to 0.284 ml L−1 depending on the pathogen tested. The optical microscope showed serious morphological damage, including cell deformation, curling, collapse, and deficiency in mycelial or conidia cell structures treated with C. fimbriata VOCs and pure compounds. In vivo tests, C. fimbriata VOCs decreased brown rot severity in peaches, and compounds Benzaldehyde and 2-Phenylethanol could reduce peach brown rot in peaches at 60 μl L−1. The VOCs produced by C. fimbriata strain have good antifungal effects; low concentration fumigation could control peach brown rot. Its fragrance is fresh, safe, and harmless, and it is possible to replace chemical fumigants. It could be used as a potential biofumigant to control fruit postharvest transportation, storage, and food preservation. To the best of our knowledge, this is the first report on the antifungal activity and biocontrol mechanism of VOCs produced by C. fimbriata.

Loquat (Rhaphiolepis biabas, heterotypic synonym: Eriobotrya japonica) is an important edible and medicinal plant that is widely cultivated on 133 thousand hectares (recorded in 2022) in China. A stem brown rot was observed on young and old trees in Mengzi city (23°23′ N; 103°23′ E), Yunnan Province, southwest China, during October 2014 and September 2021. Incidence ranged from 20% of trees in surrounding plantations to 50% incidence of a 160 tree orchard that was the focal point of the disease survey. Circular brown lesions occurred initially on the stems and gradually covered all the epidermis of the stem, leading to irregular dents within the bark that developed a dark brown powdery appearance (Fig.1A). Larger lesions affected vascular tissues, causing diseased trees to wither and die. Diseased tissues were surface-disinfected in a 5% sodium hypochlorite solution for 3 min, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA), and incubated in the dark at 28°C. Twenty samples were collected for tissue isolation, and 11 isolates were single-spored on water agar. In culture, the colonies on PDA were white to dark-gray, velvet, with dense hyphae, diameter 7.64 cm after 5 days. After 18 days, spherical or subglobose pycnidia were developed and semi-buried in medium, their walls were thicker and dark-brown, which were black particles surrounded by gray-black hyphae. Conidiogenous cells were hyaline, cylindrical, holoblastic, slightly swollen at the base, with rounded apex. Conidia were initially hyaline and aseptate with elliptic or ovate shape, becoming dark brown with a single septate and developing longitudinal striations along thick walls at maturity. Conidia dimensions varied from 8.0 to 12.2 × 3.8 to 6.1μm (n=50) (Fig.1D). The morphological characteristics of eleven isolates were consistent with the description of Lasiodiplodia theobromae (Alves et al. 2008). Further confirmation was also determined by sequencing the internal transcribed spacer (ITS), β-tubulin genes, partial translation elongation factor-1α (TEF-1α) (White et al. 1990, Carbone et al. 1999, Glass et al.1995). The isolate LSB-1 was selected for DNA sequence analysis. Based on BLASTn analysis, ITS sequences (OM617921) had 98.3% similarity with L. theobromae CBS164.96 (accession AY640255), CBS124.13(accession DQ458890), CAA006 (accession DQ458891) and CBS111530 (accession EF622074), β-tubulin sequences (OM643838) showed 99.1% similarity with L. theobromae accessions EU673110. The TEF-1α (OM643839) had 99.0% identity with L. theobromae accession EF633054. The isolate LSB-1 clustered on the same clade with other L. theobromae. Pathogenicity testing of isolate LSB-1, LSB-2, LSB-3 was conducted by inoculating the stems of l-year-old seedlings growing in pots. The epidermis at the inoculation site, 15-20 cm below the crown, was wiped with 75% alcohol cotton ball, washed three times with sterile water, and then punctured (5mm diameter) with sterile inoculation needle. A 5mm block of each isolate cultured on PDA for seven days was attached to the inoculation site. Controls were inoculated with sterile PDA blocks. The inoculation area was covered with polyethylene cling film. All inoculated seedlings were kept in controlled greenhouse at 27°C with 80% relative humidity under natural daylight conditions, and watered weekly. Each treatment was repeated three times. Eight days after inoculation, all diseased plants showed dark brown discoloration at the point of inoculation (Fig. 1G) with the bark at the inoculation site gradually raising as the disease progressed. Thirty days after inoculation, all inoculated seedlings produced typical symptoms, whereas the control seedlings remained healthy. Fungal isolates were only recovered from symptomatic stems and were morphologically identical to L. theobromae, completing Koch's postulates. According to the relevant literature, Lasiodiplodia theobromae has a broad host range, causing numerous diseases, including canker and dieback of branch (Aguilera-Cogley et al., 2021), panicle blight (Mahadevakumar et al, 2022), root rot (Abd-El Ghani and Fatouh, 2005), fruit rot(Freire et al., 2011) in diverse geographical regions. To our knowledge, this is the first report of L. theobromae causing stem brown rot of loquat in China and provides a foundation for further study of the epidemiology and integrated management of this disease.

Pear (Pyrus pyrifolia (Burm.f.) Nakai) is widely planted in China and plays a key role in economy. In the autumn of 2016, five pear fruits showing symptoms of brown rot (Fig. 1A) were found in a Suancun farmer market in Kunming, Yunnan Province, China (25°02′ N; 102°42′ E). The incidence of this disease in postharvest pear fruits ranged from 2 % to 5 % in this city. Three fruit samples were taken to run further tests. The decayed area of the fruit was soft, brown, slightly sunken, and circular. Carrot baiting was used to isolate the pathogen from symptomatic tissue (Moller et al. 1968). Primary isolates were made by transferring ascospore drops from the tips of the perithecia formed on the carrot discs onto PDA plates. Single ascospore cultures were generated by transferring single ascospores to potatoe dextrose agar (PDA) plates. Cultures were incubated 7 days at 25°C with a 12-h light/12-h dark cycle. In culture, mycelium was initially white, turned to a shallow celadon and gradually to grey-greenish later. Measurements were made 10 days after the formation of perithecia. Six pure cultures (lik-1~lik-6) were stored at -80 °C in 15% glycerol and stored at the State Key Laboratory for Conservation and Utilization of Bio-Resources of Yunnan Agricultural University. Four isolates (lik-1~lik-4) produced ascomatal bases that were submerged in the agar. Bases (Fig. 1E) were globose, black, 192.15 to 250.81 µm wide, 192.94 to 251.31 µm long, and had straight necks terminating in ostiolar hyphae (Fig. 1F) that were divergent, hyaline, and 74.19 to 116.33 µm long. Asci were not observed. Ascospores (Fig. 1I) were ovoid, hat-shaped (dimensions 3.2 to 5.1 × 2.3 to 4.6 µm). Conidiogenous cells were with enteroblastic conidium ontogeny, flask-shaped or tubular, 65.3 to 130.6 μm long, and produced cylindrical, straight aseptate conidia (8.5 to 18 × 2.5 to 3.5 µm) (Fig.1 G). All isolates produced dark brown, 10.07 to 13.08 ×8.51 to 11.64 µm aleurioconidia (Fig. 1H). Two (lik-1, lik-3) of six isolates were used for molecular identification and genomic DNA was extracted using the CTAB method (Lee & Taylor 1990). The primers ITS1 and ITS4, EF1F and EF2R were used to amplify and sequence the rDNA-ITS and TEF-1α regions (Thorpe et al. 2005; Jacobs et al. 2004). The sequences of rDNA-ITS of the isolates lik-1 and lik-3 (GenBank Accession Nos: MF153994, MF153993) showed 99.49% similarity to AF395679 (C. fimbriata isolate CMW2219). Additionally, the TEF-1α sequences of isolates lik-1 and lik-3 (GenBank Accession Nos: KY708912, KY708915) showed 100% identify to MF347676 (C. fimbriata isolate CM18). Based on symptoms, morphological characteristics, rDNA-ITS and TEF1-α sequence analysis and pathogenicity, this fungus was identified as C. fimbriata. Pathogenicity tests were conducted using 2 isolates (lik-1, lik-3) and repeated three times. Three fresh pear fruits were disinfected with 75% alcohol, then they were wounded with a 2 mm hole punch and inoculated with 200 μL conidia suspension of the fungus (approximately 2.0 × 106 conidia / mL) on the fruit surface. After inoculation pear fruits were incubated in boxes at 25°C with a relative humidity of 80% and a 12-h light / 12-h dark cycle. Three pear fruits that served as controls were wounded by punching a 2 mm hole into the skin and inoculated with 200 μL sterile distilled water. Symptoms of rot were observed one week after inoculation (Fig.1 B). The diameter of the external lesion varied from 1.5 to 2.5 cm, on average 1.9 cm. When pears were cut, the white pulp had turned black and was rotting (Fig.1 C, D). The pathogen re-isolated from all inoculated symptomatic tissue was identical to the isolates originally obtained from the pear fruits at the market by morphology and ITS analysis. No symptoms developed on the control. The pathogenicity assay showed that C. fimbriata was pathogenic on pears. To our knowledge, this is the first report of C. fimbriata on pear in China. The spread of this disease may pose a threat to pear quality in China and further studies could be performed to determine effective disease management strategies.