First Report of Root Rot Caused by Pythium dissotocum on Tobacco in China.

Tobacco (Nicotiana tabacum L.) is an important economic crop that is widely grown around the world. Its annual production in China is estimated at 2.2 million tons (Berbec and Matyka 2020). Since 2022, a root rot disease was sporadically observed on tobacco seedlings on cultivar Yunyan 87 in cultivated tobacco fields in the Hunan province of China. A disease incidence of about 10% occurred across 48 ha of tobacco fields. The affected tobacco plants had slow and stunted growth with yellowing leaves. The roots turned grayish brown, decayed, and died. Diseased roots were collected from six fields and cut into small pieces (5 mm ×5 mm) from the edge of the rotted portions, and then sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 1 min, and washed in sterilized water three times. All the sterilized tissue were placed on potato dextrose agar (PDA) medium and cultured at 26 ℃ in the dark. About 3 days later, colonies with similar morphology were removed and sub-cultured on fresh PDA. A total of six strains were obtained from six tobacco samples. Strains were white and had radial growth on PDA. Hyphae were aseptate and the sporangia were filamentous. The oogonia were subglobose, smooth, 16.04 ± 0.25 µm (n=50) in diameter, and developed on unbranched stalks. The antheridia were barrel shaped and clavate. Oospores were globose, aplerotic or nearly plerotic, measuring 6.62 ± 0.33 µm (n=50). These morphological characteristics were consistent with the description of Pythium spp. (van der Plaats-Niterink 1981). For molecular identification, the internal transcribed spacer (ITS) region of rDNA and cytochrome c oxidase subunit I (Cox I) of a representative isolate, GF-3, were amplified and sequenced (GenBank accession nos. OR228424 for ITS and OR237556 for Cox I) using universal primers ITS1/ITS4 (White et al. 1990) and FM58/FM66, respectively (Villa et al. 2006). BLASTn analysis revealed that the ITS and Cox I sequences were 99.76 % (838/840 bp) and 99.85% (671/672 bp) identical to the corresponding sequences of P. dissotocum strain CBS 166.68 (AY598634.2) and UM982 (MT981147.1), respectively. A neighbor-joining phylogenetic tree based on the Cox I sequence showed that GF-3 grouped in the P. dissotocum branch. Based on morphological and molecular characteristics, GF-3 was identified to be P. dissotocum. For pathogenicity testing, four- to five-leaf-old healthy potted tobacco seedlings of the Yunyan 87 cultivar were inoculated with a zoospore suspension (1 × 105 zoospores/ml), which was induced on V8-juice medium. The zoospore suspension was introduced into the soil around plant roots and 10 mL of inoculum was used for each plant. In the control group, plants were inoculated with sterilized water. All of the treated plants were kept in humid chambers at 26°C under a 12 h/12 h photoperiod. The pathogenicity assays were performed twice, with each treatment having three replicated plants. After 5 days, tobacco seedlings inoculated with P. dissotocum showed symptoms resembling that observed in the field. However, the control plants remained healthy. Pythium dissotocum was re-isolated from the infected plants and identified by morphological and molecular methods, thus confirming Koch's postulates. Pythium dissotocum has been reported causing root rot in other plants, including hydroponic lettuce (McGehee et al. 2018) and spinach (Huo et al. 2020). Also, many Pythium species have recently been recovered from float-bed tobacco transplant production greenhouses (Zhang et al. 2022). However, to our knowledge, this is the first report of root rot on tobacco caused by P. dissotocum in China. Since this disease could greatly affect tobacco seedling establishment in the field, appropriate management strategies need to be developed to reduce further losses in tobacco planting fields.

Characterization of a novel victorivirus from Nigrospora chinensis, a fungus isolated from tobacco.

Here, we characterized a new mycovirus from the fungus Nigrospora chinensis, which was named "Nigrospora chinensis victorivirus 1" (NcVV1). The NcVV1 genome is 5283 bp in length, containing two continuous open reading frames (ORFs), ORF1 and ORF2. ORF1 and ORF2 were predicted to encode a putative coat protein (CP) and an RNA-dependent RNA polymerase (RdRp), respectively. The stop codon of ORF1 overlaps with the start codon of ORF2 by the tetranucleotide sequence AUGA. Phylogenetic analysis based on amino acid sequences of RdRp and CP indicated that NcVV1 clustered with members of the genus Victorivirus in the family Totiviridae. To our knowledge, this was the first report of a mycovirus infecting N. chinensis.

NtMYB12a acts downstream of sucrose to inhibit fatty acid accumulation by targeting lipoxygenase and SFAR genes in tobacco.

MYB12 promotes flavonol biosynthesis in plants by targeting several early biosynthesis genes (EBGs) of this pathway. The transcriptions of these EBGs are also induced by sucrose signal. However, whether MYB12 is activated by sucrose signal and what the other roles MYB12 has in regulating plant metabolism are poorly understood. In this study, two NtMYB12 genes were cloned from Nicotiana tabacum. Both NtMYB12a and NtMYB12b are involved in regulating flavonoids biosynthesis in tobacco. NtMYB12a is further shown to inhibit the accumulation of fatty acid (FA) in tobacco leaves and seeds. Post-translational activation and chromatin immunoprecipitation assays demonstrate that NtMYB12a directly promotes the transcriptions of NtLOX6, NtLOX5, NtSFAR4 and NtGDSL2, which encode lipoxygenase (LOX) or SFAR enzymes catalyzing the degradation of FA. NtLOX6 and NtLOX5 are shown to prevent the accumulation of FA in the mature seeds and significantly reduced the percentage of polyunsaturated fatty acids (PUFAs) in tobacco. Sucrose stimulates the transcription of NtMYB12a, and loss function of NtMYB12a partially suppresses the decrease of FA content in tobacco seedlings caused by sucrose treatment. The regulation of sucrose on the expression of NtLOX6 and NtGDSL2 genes is mediated by NtMYB12a, whereas those of NtLOX5 and NtSFAR4 genes are independent of sucrose.

First Report of Fusarium commune causing Stem Rot of Tobacco (Nicotiana tabacum) in Hunan Province, China.

Tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves. It is one of the most important cash crops in China. In April of 2020, tobacco stems in commercial tobacco fields developed a brown to dark brown rot, in the Hunan Province of China. Almost 20% of the plants were infected. Symptoms appeared as round water-soaked spots, then turned dark black and developed into brown necrotic lesions leading to the stem becoming girdled and rotted. Diseased stem tissue was cut and sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 2 min, rinsed with sterile distilled water three times, and then plated on potato dextrose agar (PDA) and incubated at 26°C in the dark. Six isolates with similar morphology were obtained. Colonies cultured on PDA have morphological characteristics of Fusarium spp. producing white to orange-white, densely aerial mycelium with magenta to dark violet pigmentation. Macroconidia were produced on carnation leaf agar plates (Xi et al. 2019), which were slightly curved, with apical and basal cells curved, and usually contained three or five septa, 25.50 to 41.50×3.55 to 5.80 µm (n=50). Microconidia were cylindrical, ovate-oblong, straight to slightly curved, aseptate and 5.80 to 13.75 × 3.10 to 4.10 µm (n=50). For molecular identification, the translation elongation factor 1-alpha (EF1-α), the largest subunit of RNA polymerase II gene sequences (RPB2) and the mitochondrial small subunit rDNA (mtSSU) of a representative isolate CZ3-5-6 were amplified using the primer pairs ef1/ef2 (O'Donnell et al. 1998), 5F2/7Cr (O'Donnell et al. 2010) and NMS1/ NMS2 (Li et al. 1994). The obtained EF1-α, RPB2 and mtSSU sequences (GenBank accession nos. MT708482, MT708483 and MW260121, respectively) were 99.70 %, 100% and 100% identical to strains of F. commune (HM057338.1 for EF1-α, KU171700.1 for RPB2 and MG846025 for mtSSU). Moreover, Fusarium-ID database searches revealed that the EF1-α and RPB2 were 100% identical to F. commune strains (FD_01140_EF-1a and FD_02411_RPB2). Based on the morphological and molecular characteristics of the representative isolate, the fungal species was identified as F. commune. Pathogenicity testing of a representative isolate was performed by inoculating tobacco plants, which were grown for 2.5 months in a sterile pot with autoclaved soil. Each tobacco stem was injected with 20 µl of conidial suspension (105 spores/ml). Plants inoculated with sterilized water served as control. The pathogenicity tests were performed twice using three replicate plants, and all plants were kept in humid chambers (80 × 50 × 80 cm) at 26°C with a 12-h photoperiod. After 10 days, dark brown necrotic symptoms around the inoculated site, similar to those observed in natural field, were developed in all inoculated plants, whereas no symptoms were observed on the control plants. The pathogenic fungus was re-isolated from symptomatic tissue and identified as F. commune but was not recovered from the control plants. Fusarium commune has been reported to cause root rot or stalk and stem rot on some plants, such as sugarcane (Wang et al. 2018), Gentiana scabra (Guan et al. 2016) and maize (Xi et al. 2019). However, to our knowledge, this is the first report of F. commune causing stem rot on tobacco in China. Identification of F. commune as a stem rot causing pathogen might provide important insights for disease diagnosis on tobacco caused by different Fusarium species. Overall, this disease might bring a threat to tobacco production, and appropriate control measures should be adopted to reduce losses in tobacco fields.

Broad-spectrum antimicrobial activity and high stability of Trichokonins from Trichoderma koningii SMF2 against plant pathogens.

Antimicrobial metabolites produced by Trichoderma koningii SMF2 exhibited antimicrobial activity against a range of Gram-positive bacterial and fungal phytopathogens. Purification of these metabolites was achieved using combinations of gel filtration and high-performance liquid chromatography. Identified by liquid chromatography electrospray ionization tandem mass spectrometry, the active metabolites proved to be three known peptaibols: Trichokonin VI, VII and VIII. The Trichokonins were stable and remained biological active over a wide pH range and at every temperature tested, showing no loss of activity even after autoclaving. Trichokonins were insensitive to proteolytic enzymes. Trichokonin VI takes on typical helical structure and the structure changes only slightly at different temperatures and pH values. The present study presented the potential of Trichokonins to be used as biological control agents.