Multi-omics analysis of Streptomyces djakartensis strain MEPS155 reveal a molecular response strategy combating Ceratocystis fimbriata causing sweet potato black rot.

To investigate the potential antifungal mechanisms of rhizosphere Actinobacteria against Ceratocystis fimbriata in sweet potato, a comprehensive approach combining biochemical analyses and multi-omics techniques was employed in this study. A total of 163 bacterial strains were isolated from the rhizosphere soil of sweet potato. Among them, strain MEPS155, identified as Streptomyces djakartensis, exhibited robust and consistent inhibition of C. fimbriata mycelial growth in in vitro dual culture assays, attributed to both cell-free supernatant and volatile organic compounds. Moreover, strain MEPS155 demonstrated diverse plant growth-promoting attributes, including the production of indole-3-acetic acid, 1-aminocyclopropane-1-carboxylate deaminase, phosphorus solubilization, nitrogen fixation, and enzymatic activities such as cellulase, chitinase, and protease. Notably, strain MEPS155 exhibited efficacy against various sweet potato pathogenic fungi. Following the inoculation of strain MEPS155, a significant reduction (P < 0.05) in malondialdehyde content was observed in sweet potato slices, indicating a potential protective effect. The whole genome of MEPS155 was characterized by a size of 8,030,375 bp, encompassing 7234 coding DNA sequences and 32 secondary metabolite biosynthetic gene clusters. Transcriptomic analysis revealed 1869 differentially expressed genes in the treated group that cultured with C. fimbriata, notably influencing pathways associated with porphyrin metabolism, fatty acid biosynthesis, and biosynthesis of type II polyketide products. These alterations in gene expression are hypothesized to be linked to the production of secondary metabolites contributing to the inhibition of C. fimbriata. Metabolomic analysis identified 1469 potential differently accumulated metabolites (PDAMs) when comparing MEPS155 and the control group. The up-regulated PDAMs were predominantly associated with the biosynthesis of various secondary metabolites, including vanillin, myristic acid, and protocatechuic acid, suggesting potential inhibitory effects on plant pathogenic fungi. Our study underscores the ability of strain S. djakartensis MEPS155 to inhibit C. fimbriata growth through the production of secretory enzymes or secondary metabolites. The findings contribute to a theoretical foundation for future investigations into the role of MEPS155 in postharvest black rot prevention in sweet potato.

Cross-Inoculation of Commercial Forestry, Amenity, and Horticulture Tree Species with Ceratocystis Isolates Collected from Different Host Species.

Ceratocystis manginecans has caused significant losses in forestry productivity in Indonesia and neighboring nations. It also infects horticultural trees, but the host range of individual isolates of C. manginecans is poorly studied. So, this study aimed to better understand the potential host range and evaluate aggressiveness against forestry and fruit tree species of C. manginecans isolated from various tree species in Indonesia. Five C. manginecans isolates, four from different tree species and one from the shot-hole borer Euwallacea perbrevis, were used to inoculate seven fruit and six forest tree species, including E. pellita and Acacia mangium. Many of the inoculated trees produced typical canker disease symptoms, such as rough, swollen, and cracked lesions on the bark, but some trees did not have any external symptoms. Mortality in the most susceptible clone of A. mangium was 40% within 8 weeks. Forest tree species were more susceptible than fruit trees, with the length of xylem discoloration ranging from 0.4 to 101 cm. In fruit trees, the average extent of xylem discoloration was lower, ranging from 0.4 to 20.5 cm; however, mortalities were recorded in two fruit tree species, Citrus microcarpa and Durio zibethinus. Host-isolate interaction was evident; isolate Ep106C from Eucalyptus pellita caused the greatest xylem discoloration in Citrus sp., whereas Hy163C from Hymenaea courbaril was the most damaging in D. zibethinus, Artocarpus heterophyllus, and Mangifera indica. Increasingly globalized food and fiber systems increase risk of disease spread, and the serious threat of C. manginecans incursions into countries where it is not present must be evaluated more thoroughly.

Drosophila hydei as a Potential Vector of Ceratocystis fimbriata, the Causal Agent of Sweetpotato Black Rot, in Storage Facilities.

Ceratocystis fimbriata, the causal agent of sweetpotato black rot, is a pathogen capable of developing and spreading within postharvest settings. A survey of North Carolina sweetpotato storage facilities was conducted to determine the arthropods present and identify potential vectors of C. fimbriata. Sixteen taxonomic categories were recovered, and the genus Drosophila (Diptera: Drosophilidae) accounted for 79% of individuals sampled, with Drosophila hydei being the most abundant species. Behavioral assays were conducted to determine if D. hydei is attracted to C. fimbriata-inoculated roots and if the pathogen could be recovered from external or internal surfaces of the insect. Flies were released in insect-trapping pitchers containing either C. fimbriata-inoculated or noninoculated roots or Petri dishes. No significant differences in fly number were detected in sweetpotato-baited pitchers; however, significant differences were found in the pitcher baited with a mature C. fimbriata culture. Flies were subjected to washes to determine if viable C. fimbriata was present (internally or externally); washes were plated onto carrot agar plates and observed for the presence of C. fimbriata colonies. Both external and internal washes had viable C. fimbriata inocula with no significant differences, and inoculated sweetpotatoes had a significantly higher number of flies carrying C. fimbriata. This study suggests that D. hydei can carry C. fimbriata from infected sweetpotatoes and move viable C. fimbriata inocula both externally and internally, making this the first report of any Drosophila sp. serving as a potential vector for the Ceratocystis genus.

Effector Repertoire of the Sweetpotato Black Rot Fungal Pathogen Ceratocystis fimbriata.

In 2015, sweetpotato producers in the United States experienced one of the worst outbreaks of black rot recorded in history, with up to 60% losses reported in the field and packing houses and at shipping ports. Host resistance remains the ideal management tool to decrease crop losses. Lack of knowledge of Ceratocystis fimbriata biology represents a critical barrier for the deployment of resistance to black rot in sweetpotato. In this study, we scanned the recent near chromosomal-level assembly for putative secreted effectors in the sweetpotato C. fimbriata isolate AS236 using a custom fungal effector annotation pipeline. We identified a set of 188 putative effectors on the basis of secretion signal and in silico prediction in EffectorP. We conducted a deep RNA time-course sequencing experiment to determine whether C. fimbriata modulates effectors in planta and to define a candidate list of effectors expressed during infection. We examined the expression profile of two C. fimbriata isolates, a pre-epidemic (1990s) isolate and a post-epidemic (2015) isolate. Our in planta expression profiling revealed clusters of co-expressed secreted effector candidates. Based on fold-change differences of putative effectors in both isolates and over the course of infection, we suggested prioritization of 31 effectors for functional characterization. Among this set, we identified several effectors that provide evidence for a marked biotrophic phase in C. fimbriata during infection of sweetpotato storage roots. Our study revealed a catalog of effector proteins that provide insight into C. fimbriata infection mechanisms and represent a core catalog to implement effector-assisted breeding in sweetpotato. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Novel Multiresistant Osmotin-like Protein from Sweetpotato as a Promising Biofungicide to Control Ceratocystis fimbriata by Destroying Spores through Accumulation of Reactive Oxygen Species.

Osmotin-like proteins (OLPs) play an important role in host-plant defense. In this study, a novel multiresistant OLP (IbOLP1) was screened from sweetpotato (Ipomoea batatas) with a molecular weight of 26.3 kDa. The expression level of IbOLP1 was significantly higher in resistant cultivars than susceptible ones after inoculation with Ceratocystis fimbriata, which causes black rot disease in sweetpotato. The expression of IbOLP1 in Pichia pastoris led to the lysis of yeast cells themselves. The recombinant IbOLP1 displayed antifungal, antibacterial, and antinematode activity and stability. IbOLP1 could restrain the mycelial growth and lyse spores of C. fimbriata, distinctly reducing the incidence of black rot in sweetpotato. The IbOLP1 can trigger the apoptosis of black rot spores by elevating the intracellular levels of reactive oxygen species. Collectively, these findings suggest that IbOLP1 can be used to develop natural antimicrobial resources instead of chemical agents and generate new, disease-resistant germplasm.

The APSES transcription factor CfSwi6 is required for growth, cell wall integrity, and pathogenicity of Ceratocystis fimbriata.

Cell wall integrity (CWI) is crucial for the growth, development, and host invasion of pathogenic fungi. The APSES transcription factor Swi6 in fungi plays a role in mediating cell wall integrity through the mitogen-activated protein kinase (MAPK) signaling pathway. Ceratocystis fimbriata is a notorious pathogenic fungus responsible for causing black rot in sweet potatoes. In this study, an orthologous APSES transcription factor Swi6 (CfSwi6) downstream of the CWI regulatory pathway in C. fimbriata was characterized. Deletion of CfSWI6 leads to impaired hyphal development, conidiation, and compromised cell wall integrity, resulting in a significant reduction in virulence. Transcriptome analysis revealed the involvement of CfSWI6 in various pathways, including the MAPK pathway, DNA synthesis and stress response. ChIP-seq data provided predictions of potential target genes regulated by CfSwi6. Through yeast one-hybrid, we confirmed the direct binding of CfSwi6 to the promoter of the chitin synthetase gene. In summary, these findings indicated that CfSwi6 plays an important role in the growth, development, and pathogenicity of C. fimbriata. This study provides new insights into the pathogenic mechanism of C. fimbriata in sweet potato and inspires potential strategies to control sweet potato black rot.

Long-Read Sequencing Genome Assembly of Ceratocystis fimbriata Enables Development of Molecular Diagnostics for Sweetpotato Black Rot.

Ceratocystis fimbriata is a destructive fungal pathogen of sweetpotato (Ipomoea batatas) that leads to losses at all stages of sweetpotato production. Accurate detection of C. fimbriata would allow for more efficient deployment of management tactics in sweetpotato production. To develop a diagnostic assay, a hybrid genome assembly of C. fimbriata isolate AS236 was generated. The resulting 31.7-MB assembly was near-chromosome level, with 18 contigs, 6,481 predicted genes, and a BUSCO completion score of 98.4% when compared with the fungus-specific lineage database. Additional Illumina DNA reads from C. manginecans, C. platani, and a second C. fimbriata isolate (C1421) were then mapped to the assembled genome using BOWTIE2 and counted using HTSeq, which identified 148 genes present only within C. fimbriata as molecular diagnostic candidates; 6 single-copy and 35 highly multi-copy (>40 BLAST hits), as determined through a self-BLAST-P alignment. Primers for PCR were designed in the 200-bp flanking region of the first exon for each candidate, and the candidates were validated against a diverse DNA panel containing Ceratocystis species, sweetpotato pathogens, and plants. After validation, two diagnostic candidates amplified only C. fimbriata DNA and were considered to be highly specific to the species. These genetic markers will serve as valuable diagnostic tools with multiple applications including the detection of C. fimbriata in seed, soil, and wash water in sweetpotato production.

CfErp3 regulates growth, conidiation, inducing ipomeamarone and the pathogenicity of Ceratocystis fimbriata.

The Erp3 protein, which is an important member of the p24 family, is primarily responsible for the transport of cargo from the ER to the Golgi apparatus in Saccharomyces cerevisiae. However, the function of Erp3 in plant pathogenic fungi has not been reported. In this study, we characterized the ERP3 gene in Ceratocystis fimbriata, which causes the devastating disease sweetpotato black rot. The ΔCferp3 mutants exhibited slow growth, reduced conidia production, attenuated virulence, and reduced ability to induce host to produce toxins. Further analysis revealed that CfErp3 was localized in the ER and vesicles and regulated endocytosis, cell wall integrity, and osmotic stress responses, modulated ROS levels, and the production of ipomeamarone during pathogen-host interactions. These results indicate that CfErp3 regulates C. fimbriata growth and pathogenicity as well as the production of ipomeamarone in sweetpotato by controlling endocytosis, oxidative homeostasis, and responses to cell wall and osmotic stresses.

The Mechanism of Transcription Factor Swi6 in Regulating Growth and Pathogenicity of Ceratocystis fimbriata: Insights from Non-Targeted Metabolomics.

Ceratocystis fimbriata (C. fimbriata) is a notorious pathogenic fungus that causes sweet potato black rot disease. The APSES transcription factor Swi6 in fungi is located downstream of the cell wall integrity (CWI)-mitogen-activated protein kinase (MAPK) signaling pathway and has been identified to be involved in cell wall integrity and virulence in several filamentous pathogenic fungi. However, the specific mechanisms by which Swi6 regulates the growth and pathogenicity of plant pathogenic fungi remain elusive. In this study, the SWI6 deletion mutants and complemented strains of C. fimbriata were generated. Deletion of Swi6 in C. fimbriata resulted in aberrant growth patterns. Pathogenicity assays on sweet potato storage roots revealed a significant decrease in virulence in the mutant. Non-targeted metabolomic analysis using LC-MS identified a total of 692 potential differentially accumulated metabolites (PDAMs) in the ∆Cfswi6 mutant compared to the wild type, and the results of KEGG enrichment analysis demonstrated significant enrichment of PDAMs within various metabolic pathways, including amino acid metabolism, lipid metabolism, nucleotide metabolism, GPI-anchored protein synthesis, and ABC transporter metabolism. These metabolic pathways were believed to play a crucial role in mediating the growth and pathogenicity of C. fimbriata through the regulation of CWI. Firstly, the deletion of the SWI6 gene led to abnormal amino acid and lipid metabolism, potentially exacerbating energy storage imbalance. Secondly, significant enrichment of metabolites related to GPI-anchored protein biosynthesis implied compromised cell wall integrity. Lastly, disruption of ABC transport protein metabolism may hinder intracellular transmembrane transport. Importantly, this study represents the first investigation into the potential regulatory mechanisms of SWI6 in plant filamentous pathogenic fungi from a metabolic perspective. The findings provide novel insights into the role of SWI6 in the growth and virulence of C. fimbriata, highlighting its potential as a target for controlling this pathogen.

Ceratocystis fimbriata Causing Postharvest Fruit Rot of Pear in Punjab, Pakistan.

Pear (Pyrus communis) is an important deciduous fruit cultivated on a worldwide scale including Pakistan. During August 2021, a postharvest fruit rot disease was observed on several pears at various farmers market in Okara- a district of Punjab Province, Pakistan. The incidence of the disease varied from 7 to 20% with 35% disease severity. Necrotic spots (10 to 20 mm diameter) were first observed on the infected pear fruit. The spots enlarged gradually and developed into a brown, water-soaked and rotted lesion. Eventually, the whole fruit became soft, rotted and covered with a gray-brown mycelium. The isolates were obtained from the symptomatic tissues (n = 18) incubated on carrot discs that had been surface sterilized in 100-ppm streptomycin solution. After consistent sporulation of a fungus on the carrot discs, the ascospore masses formed at the tip of perithecia were transferred to malt extract agar (MEA). Primary conidia were cylindrical and hyaline (7 to 11 × 4 to 7 µm) and secondary conidia were hyaline and barrel-shaped (7 to 12 × 5 to 8 µm). Endoconidiophores with primary conidia were (12 to 27 × 2.6 to 5.5 µm). Perithecia produced on carrot discs were dark brown to black, and the base was 157 to 278 µm in diameter. Ascomatal necks were 512 to 656 µm long, dark brown to black, lighter in color at apices, tapering from base (23 to 45 µm diameter) to apex (13 to 24 µm diameter). Ostiolar hyphae were 41 to 79 µm long. Ascospores were hyaline, hat shaped, 3 to 4 µm long, and accumulated in a sticky matrix at the tips of perithecial necks. Mycelium was initially hyaline but became dark greenish brown after 7 days. Dark brown, thick-walled aleuroconidia (13 to 19.5 × 9 to 14 µm) appeared on culture plates after 2 months. Based on morphological characteristics, the fungus was identified as Ceratocystis fimbriata (Engelbrecht, 2005; Suwandi et al. 2021). To further confirm species identification, genomic DNA of two representative isolates (UO-05 and UO-06) was obtained using an extraction kit. The internal transcribed spacer (ITS) region was amplified using ITS1/4 (White et al. 1990). A BLAST search with GenBank accession nos. OR185451 and OR185456 indicated 99 to 100% identity with several C. fimbriata including type species (MH856050.1; KC493160.1; MT560374.1). Pathogenicity tests were conducted by inoculating nine disease-free pear (cv. Concord) fruit after disinfesting in 75% ethanol. A prepared spore suspension (1.0 × 106 spores/ml) was dropped on the wounds (a depth of 1 mm diameter) on the pear surface, which were made by a sterilized needle. 10 µl of a prepared spore suspension was dropped onto nine pears. Sterile water (10 µl) was dropped on the wounded sites of nine pear fruits as negative controls and all fruits were incubated in a growth chamber at 30/26°C (day/night, 90% relative humidity). Symptoms similar to those on the naturally infected fruits began after 4 to 5 days of inoculation, while controls remained healthy. The fungal isolates recovered from inoculated pears were morphologically identical to the C. fimbriata isolates originally recovered from symptomatic fruits fulfilling Koch's postulates. The pathogen has been reported to cause postharvest fruit rot of passion fruit and cucumber (Firmino et al. 2016; Li et al. 2019). To our knowledge, this is the first report of C. fimbriata causing fruit rot of pear in Punjab Province. The detection of this disease will help pear growers to take actions to monitor and prevent disease outbreak as well as develop an effective management practice when it occurs.

Molecular Characterization and Genetic Variation in Ceratocystis fimbriata Ell. and Halst. on Pomegranate

Fifteen isolates of Ceratocystis fimbriata collected from different locations in Karnataka were characterized using ITS gene technology. It produced an amplification size of 600−650 bp, which indicated that all the isolates belong to the genus Ceratocystis, thus confirming the identity of the pathogenic isolates. To test genetic variability, isolates were analyzed using microsatellite markers. An UPGMA dendrogram for genetic variation among the isolates showed that all the isolates fell into two major clusters. The first cluster consisted of isolate Cf-10 and the second cluster was further divided into two sub-clusters. Sub-cluster one consisted of isolate Cf-2. Sub-cluster two was again divided into five groups. The first group included isolate Cf-13, the second group consisted of isolate Cf-14, the third group included isolates Cf-1, Cf-4, Cf-6, Cf-7, Cf-8 and Cf-9, the fourth group included Cf-5 and Cf-11, and the fifth group consisted of Cf-3, Cf-12 and Cf-15. The dissimilarity coefficient ranged from 0.00 to 0.20 among the isolates. Isolates Cf-1, Cf-3, Cf-4, Cf-5 Cf-6, Cf-7, Cf-8, Cf-9, Cf-11, Cf-12 and Cf-15 were found to be highly similar, as their dissimilarity coefficient was zero. Maximum dissimilarity (0.20) was found between isolate Cf-10 and all the other isolates, suggesting they were genetically distinct.

Antifungal activity of the volatile organic compounds produced by Ceratocystis fimbriata strains WSJK-1 and Mby.

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.

Diversity of endophytic fungi isolated from different plant parts of Acacia mangium, and antagonistic activity against Ceratocystis fimbriata, a causal agent of Ceratocystis wilt disease of A. mangium in Malaysia.

Acacia mangium is an important wood for commercial products especially pulp and medium-density fibreboard. However, it is susceptible to Ceratocystis fimbriata infection, leading to Ceratocystis wilt. Therefore, the present work aimed to (i) establish the diversity of endophytic fungi in different plant parts of A. mangium,and (ii) evaluate the antifungal potentials of the isolated and identified endophytic fungi against C. fimbriata. Endophytic fungal identification was conducted by PCR amplification and sequencing of the internal transcribed spacer 1 (ITS1) and ITS4 regions of nuclear ribosomal DNA. A total of 66 endophytic fungi were successfully isolated from different parts of A. mangium; leaf (21), stem (13), petiole (12), root (9), flower (6), and fruit (5). The endophytic fungal isolates belonged to Ascomycota (95.5%) and Zygomycota (4.5%). For Ascomycota 13 genera were identified: Trichoderma (28.6%), Nigrospora (28.6%), Pestalotiopsis (12.7%), Lasiodiplodia (9.5%), Aspergillus (6.3%), Sordariomycetes (3%), and Neopestalotiopsis, Pseudopestalotiopsis, Eutiarosporella, Curvularia, Fusarium, Penicillium, and Hypoxylon each with a single isolate. For Zygomycota, only Blakeslea sp. (5%) was isolated. Against C. fimbriata, Trichoderma koningiopsis (AC 1S) from stem, Nigrospora oryzae (AC 7L) from leaf, Nigrospora sphaerica (AC 3F) from the flower, Lasiodiplodia sp. (AC 2 U) from fruit, Nigrospora sphaerica (AC 4P) from petiole, and Trichoderma sp. (AC 9R) from root exhibited strong inhibition for C. fimbriata between 58.33 to 69.23%. Thus, it can be concluded that certain endophytic fungi of A. mangium have the potential to be harnessed as anti-Ceratocystis agent in future biotechnological applications.

First Report of a New Postharvest Disease of Pear Fruit Caused by Ceratocystis fimbriata in Kunming, China.

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.

Antifungal Volatile Organic Compounds from Streptomyces setonii WY228 Control Black Spot Disease of Sweet Potato.

Volatile organic compounds (VOCs) produced by microorganisms are considered promising environmental-safety fumigants for controlling postharvest diseases. Ceratocystis fimbriata, the pathogen of black spot disease, seriously affects the quality and yield of sweet potato in the field and postharvest. This study tested the effects of VOCs produced by Streptomyces setonii WY228 on the control of C. fimbriata in vitro and in vivo. The VOCs exhibited strong antifungal activity and significantly inhibited the growth of C. fimbriata. During the 20-day storage, VOC fumigation significantly controlled the occurrence of the pathogen, increased the content of antioxidants and defense-related enzymes and flavonoids, and boosted the starch content so as to maintain the quality of the sweet potatoes. Headspace analysis showed that the volatiles 2-ethyl-5-methylpyrazine and dimethyl disulfide significantly inhibited the mycelial growth and spore germination of C. fimbriata in a dose-dependent manner. Fumigation with 100 µL/L 2-ethyl-5-methylpyrazine completely controlled the pathogen in vivo after 10 days of storage. Transcriptome analysis showed that volatiles mainly downregulated the ribosomal synthesis genes and activated the proteasome system of the pathogen in response to VOC stress, while the genes related to spore development, cell membrane synthesis, mitochondrial function, and hydrolase and toxin synthesis were also downregulated, indicating that WY228-produced VOCs have diverse modes of action for pathogen control. Our study demonstrates that fumigation of sweet potato tuberous roots with S. setonii WY228 or use of formulations based on the VOCs is a promising new strategy to control sweet potato and other food and fruit pathogens during storage and shipment. IMPORTANCE Black spot disease caused by Ceratocystis fimbriata has caused huge economic losses to worldwide sweet potato production. At present, the control of C. fimbriata mainly depends on toxic fungicides, and there is a lack of effective alternative strategies. The research on biological control of sweet potato black spot disease is also very limited. An efficient biocontrol technique against pathogens using microbial volatile organic compounds could be an alternative method to control this disease. Our study revealed the significant biological control effect of volatile organic compounds of Streptomyces setonii WY228 on black spot disease of postharvest sweet potato and the complex antifungal mechanism against C. fimbriata. Our data demonstrated that Streptomyces setonii WY228 and its volatile 2-ethyl-5-methylpyrazine could be a candidate strain and compound for the creation of fumigants and showed the important potential of biotechnology applications in the field of food and agriculture.

Effect of tebuconazole and trifloxystrobin on Ceratocystis fimbriata to control black rot of sweet potato: processes of reactive oxygen species generation and antioxidant defense responses.

Black rot, caused by Ceratocystis fimbriata, is one of the most destructive disease of sweet potato worldwide, resulting in significant yield losses. However, a proper management system can increase resistance to this disease. Therefore, this study investigated the potential of using tebuconazole (TEB) and trifloxystrobin (TRI) to improve the antioxidant defense systems in sweet potato as well as the inhibitory effects on the growth of and antioxidant activity in C. fimbriata. Four days after inoculating cut surfaces of sweet potato disks with C. fimbriata, disease development was reduced by different concentrations of TEB + TRI. Infection by C. fimbriata increased the levels of hydrogen peroxide (H2O2), malondialdehyde (MDA), and electrolyte leakage (EL), and the activity of lipoxygenase (LOX) by 138, 152, 73, and 282%, respectively, in sweet potato disks, relative to control. In the sweet potato disks, C. fimbriata reduced the antioxidant enzyme activities as well as the contents of ascorbate (AsA) and reduced glutathione (GSH) by 82 and 91%, respectively, compared with control. However, TEB + TRI reduced the oxidative damage in the C. fimbriata-inoculated sweet potato disks by enhancing the antioxidant defense systems. On the other hand, applying TEB + TRI increased the levels of H2O2, MDA, and EL, and increased the activity of LOX in C. fimbriata, in which the contents of AsA and GSH decreased, and therefore, inhibited the growth of C. fimbriata. These results suggest that TEB + TRI can significantly control black rot disease in sweet potato by inhibiting the growth of C. fimbriata.

Mode of action and efficacy of quinolinic acid for the control of Ceratocystis fimbriata on sweet potato.

BACKGROUND: Ceratocystis fimbriata is a hazardous fungal pathogen able to cause black rot disease on sweet potato. The management of C. fimbriata strongly relies on the use of toxic fungicides, and there is a lack of efficient alternative strategies. RESULTS: The antifungal properties of quinolinic acid (QA) were studied for the first time, indicating that QA shows selective antifungal activity against C. fimbriata. QA inhibited completely the mycelial growth of C. fimbriata at less than 0.8 mg mL-1 concentration (pH 4), and was able to produce alterations in the fungal cell wall, and to impede spore agglutination and mycelium formation. QA significantly reduced the concentration of ergosterol, and was able to associate to iron (II), suggesting that QA may be a lanosterol 14-α demethylase inhibitor. In preventive applications, QA reduced the disease incidence of C. fimbriata on sweet potato by 75%, achieving higher control efficacy in comparison with commercial fungicides prochloraz and carbendazim. CONCLUSIONS: The first selective antifungal agent against C. fimbriata was discovered in this work, and showed suitable antifungal properties for the management of black rot disease. © 2021 Society of Chemical Industry.

Identification and Characterization of Ceratocystis fimbriata Causing Lethal Wilt on the Lansium Tree in Indonesia.

Bark canker, wood discoloration, and wilting of the duku tree (Lansium domesticum) along the watershed of Komering River, South Sumatra Province, Indonesia first appeared in 2013. The incidence of tree mortality was 100% within 3 years in badly infected orchards. A Ceratocystis species was consistently isolated from the diseased tissue and identified by morphological and sequence analyses of the internal transcribed spacer (ITS) and ß-tubulin regions. Pathogenicity tests were conducted and Koch's postulates were confirmed. The fungus was also pathogenic on Acacia mangium, but was less pathogenic on mango. Partial flooding was unfavourable for disease development. Two described isolates (WRC and WBC) had minor variation in morphology and DNA sequences, but the former exhibited a more pathogenic on both duku and acacia. The ITS phylogenies grouped the most pathogenic isolate (WRC) causing wilting of the duku tree within the aggressive and widely distributed ITS5 haplotype of C. fimbriata.