First Report of Tobacco Leaf Tip Blight Caused by Alternaria gossypina in China.

Tobacco (Nicotiana tabacum L.) belongs to the family Solanaceae, an economically significant crop (Zhou et al. 2023). Twelve samples with leaf spots were collected in Keti Village, Changshun County, Zunyi City, Guizhou province, China in 2022. Twenty-five percent of the samples had dry lesions near the leaf tip which resulted leaf tip blight after development. Fungi were isolated by a previous method (Wei et al. 2022). Six Alternaria strains were obtained and preserved in the Fungal Herbarium of Yangtze University (YZU), Jingzhou, Hubei, China. Among them, one strain YZU 221477 showed distinct cultural characteristics out of five A. alternata strains, which was again determined by growing on potato dextrose agar (PDA) at 25°C for 7 days in dark to evaluate. The colonies (60 mm in diameter) were white cottony in the center surrounded by vinaceous purple. To examine the morphology, mycelia were inoculated onto potato carrot agar (PCA) at 22°C, following an 8 h light/16 h dark photoperiod (Simmons 2007). Conidia were obclavate or ovoid, normally 3-5 conidial units per chain, 20-38 × 10-16.5 µm, 3 to 5 transverse septa, beakless or a short beak (4-30 µm). The observation results were consistent with those of A. gossypina (Zhang 2003). Total genomic DNA was extracted using the CTAB method and seven gene regions including internal transcribed spacer of rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1 alpha (TEF1), RNA polymerase second largest subunit (RPB2), Alternaria major allergen gene (Alt a 1), endopolygalacturonase (EndoPG) and an anonymous gene region (OPA10-2) were amplified with ITS5/ITS4, gpd1/gpd2, EF1-728F/EF1-986R, RPB2-5F/RPB2-7cR, Alt-for/Alt-rev, PG3/PG2b and OPA10-2L/OPA10-2R primers, respectively. All sequences were deposited in GenBank (ITS: OR710806; GAPDH: PP057862; TEF1: PP158601; RPB2: PP057863; Alt a 1: PP057865; EndoPG: PP057861; OPA10-2: PP057864). Combining with relevant sequences retrieved from the NCBI database were used for the phylogenetic analysis. Maximum Likelihood (ML) tree was constructed with RAxML v.7.2.8 employing GTRCAT model using 1000 bootstrap (BS) replicates to assess statistical support. The results indicated that the present strain grouped with A. gossypina (type strain of CBS 104.32) supported with 73% bootstrap values, also having a support of 0.83 Bayesian posterior probabilities values. Based on morphology and molecular evidence, the strain YZU 221477 is identified as Alternaria gossypina. Pathogenicity was examined to fulfill Koch's postulates. Mycelial plugs (6 mm diameter) of the present strain and A. alternata cultivated on PDA were taken from the margin and inoculated onto viable tobacco leaves (Cultivar: Yunyan 87, n=3) growing forty days, while controls were inoculated with sterile PDA. The assay was conducted three times. The plants were maintained at 25°C with humidity levels over 85% in a greenhouse. Leaves were evaluated after 7 days, necrotic spots encircled by yellow halos were on both inoculums, except controls. Pathogen re-isolation confirmed that it was the same as inoculated fungus based on morphology. A. gossypina was firstly found on cotton (Hopkins 1931), late reported to induce disease on Minneola, Nopalea, Hibiscus, Citrus, Solanum and Ageratina. To our knowledge, this is the first report of A. gossypina causing tobacco leaf tip blight in China, and it also provides a basis for controlling of tobacco leaf tip blight.

Characteristics of Epicoccum latusicollum as revealed by genomic and metabolic phenomic analysis, the causal agent of tobacco Epicoccus leaf spot.

Epicoccum latusicollum is a fungus that causes a severe foliar disease on flue-cured tobacco in southwest China, resulting in significant losses in tobacco yield and quality. To better understand the organism, researchers investigated its optimal growth conditions and metabolic versatility using a combination of traditional methods and the Biolog Phenotype MicroArray technique. The study found that E. latusicollum exhibited impressive metabolic versatility, being able to metabolize a majority of carbon, nitrogen, sulfur, and phosphorus sources tested, as well as adapt to different environmental conditions, including broad pH ranges and various osmolytes. The optimal medium for mycelial growth was alkyl ester agar medium, while oatmeal agar medium was optimal for sporulation, and the optimum temperature for mycelial growth was 25°C. The lethal temperature was 40°C. The study also identified arbutin and amygdalin as optimal carbon sources and Ala-Asp and Ala-Glu as optimal nitrogen sources for E. latusicollum. Furthermore, the genome of E. latusicollum strain T41 was sequenced using Illumina HiSeq and Pacific Biosciences technologies, with 10,821 genes predicted using Nonredundant, Gene Ontology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes, and SWISS-PROT databases. Analysis of the metabolic functions of phyllosphere microorganisms on diseased tobacco leaves affected by E. latusicollum using the Biolog Eco microplate revealed an inability to efficiently metabolize a total of 29 carbon sources, with only tween 40 showing some metabolizing ability. The study provides new insights into the structure and function of phyllosphere microbiota and highlights important challenges for future research, as well as a theoretical basis for the integrated control and breeding for disease resistance of tobacco Epicoccus leaf spot. This information can be useful in developing new strategies for disease control and management, as well as enhancing crop productivity and quality.

Variations in leaf phyllosphere microbial communities and development of tobacco brown spot before and after fungicide application.

In recent years, STROBY (50% Kresoxim-methyl) has been widely used to control tobacco brown spot in Guizhou Province, China. As a broad-spectrum fungicide, STROBY targets not only phytopathogens, but also affects many other microorganisms including those pathogenic, beneficial, or neutral to the plant hosts. To understand the effects of STROBY on the phyllosphere microbial communities of tobacco leaves during the development of tobacco brown spot, the fungal and bacterial communities of symptomatic and asymptomatic leaves at four time points, before spraying (August 29) and after spraying (September 3, 8, and 13), were investigated using the Illumina high-throughput sequencing. The results showed that STROBY had significant effects on the phyllosphere microbial communities of tobacco leaves. Microbial communities in asymptomatic leaves were more greatly affected than their counterparts in symptomatic leaves, and fungal communities were more sensitive than bacterial communities. Throughout the experiment, the most common genera in symptomatic leaves were Alternaria, Pseudomonas, Pantoea, and Sphingomonas, and in asymptomatic leaves, these were Golubevia and Pantoea. After spraying, the alpha diversity of fungal communities increased in symptomatic leaves and decreased in asymptomatic leaves, while the alpha diversity of bacteria increased in both types of leaves. Beta diversity showed that in asymptomatic leaves, the fungal communities in the first stage was significantly different from the remaining three stages. In contrast, the fungal communities in symptomatic leaves and the bacterial communities in all leaves did not fluctuate significantly during the four stages. Before spraying (August 29), the dominant functions of the fungal community were animal pathogen, endophyte, plant pathogen, and wood saprotroph. Whereas after spraying (September 3, 8, and 13), the proportion of the above fungal functions decreased and the unassigned functions increased, especially in asymptomatic leaves. This study describes the effects of STROBY application and tobacco brown spot presence in shaping the leaf phyllosphere microbial communities, and provides insights into the microbial community effects on tobacco leaves of a strobilurin fungicide.

Spatio-Temporal Variation in the Phyllospheric Microbial Biodiversity of Alternaria Alternata-Infected Tobacco Foliage.

Phyllospheric microbial composition of tobacco (Nicotiana tabacum L.) is contingent upon certain factors, such as the growth stage of the plant, leaf position, and cultivar and its geographical location, which influence, either directly or indirectly, the growth, overall health, and production of the tobacco plant. To better understand the spatiotemporal variation of the community and the divergence of phyllospheric microflora, procured from healthy and diseased tobacco leaves infected by Alternaria alternata, the current study employed microbe culturing, high-throughput technique, and BIOLOG ECO. Microbe culturing resulted in the isolation of 153 culturable fungal isolates belonging to 33 genera and 99 bacterial isolates belonging to 15 genera. High-throughput sequencing revealed that the phyllosphere of tobacco was dominantly colonized by Ascomycota and Proteobacteria, whereas, the most abundant fungal and bacterial genera were Alternaria and Pseudomonas. The relative abundance of Alternaria increased in the upper and middle healthy groups from the first collection time to the third, whereas, the relative abundance of Pseudomonas, Sphingomonas, and Methylobacterium from the same positions increased during gradual leaf aging. Non-metric multi-dimensional scaling (NMDs) showed clustering of fungal communities in healthy samples, while bacterial communities of all diseased and healthy groups were found scattered. FUNGuild analysis, from the first collection stage to the third one in both groups, indicated an increase in the relative abundance of Pathotroph-Saprotroph, Pathotroph-Saprotroph-Symbiotroph, and Pathotroph-Symbiotroph. Inclusive of all samples, as per the PICRUSt analysis, the predominant pathway was metabolism function accounting for 50.03%. The average values of omnilog units (OUs) showed relatively higher utilization rates of carbon sources by the microbial flora of healthy leaves. According to the analysis of genus abundances, leaf growth and leaf position were the important drivers of change in structuring the microbial communities. The current findings revealed the complex ecological dynamics that occur in the phyllospheric microbial communities over the course of a spatiotemporal varying environment with the development of tobacco brown spots, highlighting the importance of community succession.

Analysis of Phyllosphere Microorganisms and Potential Pathogens of Tobacco Leaves.

In the tobacco phyllosphere, some of the microbes may have detrimental effects on plant health, while many may be neutral or even beneficial. Some cannot be cultivated, so culture-independent methods are needed to explore microbial diversity. In this study, both metagenetic analysis and traditional culture-dependent methods were used on asymptomatic healthy leaves and symptomatic diseased leaves of tobacco plants. In the culture-independent analysis, asymptomatic leaves had higher microbial diversity and richness than symptomatic leaves. Both asymptomatic and symptomatic leaves contained several potentially pathogenic bacterial and fungal genera. The putative bacterial pathogens, such as species of Pseudomonas, Pantoea, or Ralstonia, and putative fungal pathogens, such as species of Phoma, Cladosporium, Alternaria, Fusarium, Corynespora, and Epicoccum, had a higher relative abundance in symptomatic leaves than asymptomatic leaves. FUNGuild analysis indicated that the foliar fungal community also included endophytes, saprotrophs, epiphytes, parasites, and endosymbionts. PICRUSt analysis showed that the dominant functions of the bacterial community in a symptomatic leaf were cellular processes and environmental information processing. In the other five foliar samples, the dominant functions of the bacterial community were genetic information processing, metabolism, and organismal systems. In the traditional culture-dependent method, 47 fungal strains were isolated from 60 symptomatic tobacco leaf fragments bearing leaf spots. Among them, 21 strains of Colletotrichum (29%), Xylariaceae (14%), Corynespora (14%), Pestalotiopsis (10%), Alternaria (10%), Epicoccum (10%), Byssosphaeria (5%), Phoma (5%), and Diaporthe (5%) all fulfilled Koch's postulates and were found to cause disease on detached tobacco leaves in artificial inoculation tests. Symptoms on detached leaves caused by three strains of Corynespora cassiicola in artificial inoculation tests were similar to the original disease symptoms in the tobacco field. This study showed that the combined application of culture-dependent and independent methods could give comprehensive insights into microbial composition that each method alone did not reveal.

Phenotypic analysis and genome sequence of Rhizopus oryzae strain Y5, the causal agent of tobacco pole rot.

Rhizopus oryzae is a destructive pathogen that frequently causes tobacco pole rot in curing chambers. Phenotypic characterization of the pathogen was conducted to provide basic biological and pathological information using Biolog Phenotype MicroArray (PM). In addition, the Y5 strain of R. oryzae was sequenced using Illumina HiSeq and Pacific Biosciences (PacBio) technologies. Using PM plates 1-8, 758 growth conditions were tested. Results indicated that R. oryzae could metabolize 54.21% of tested carbon sources, 86.84% of nitrogen sources, 100% of sulfur sources, and 98.31% of phosphorus sources. About 37 carbon compounds, including D-xylose, N-acetyl-D-glucosamine, D-sorbitol, ß-methyl-D-glucoside, D-galactose, L-arabinose, and D-cellobiose, significantly supported the growth of the pathogen. PM 3 indicated the active nitrogen sources, including Gly-Asn, Ala-Asp., Ala-Gln, and uric acid. PM 6-8 showed 285 different nitrogen pathways, indicating that different combinations of different amino acids support the growth of the pathogen. Genome sequencing results showed that the R. oryzae Y5 strain had raw data assembled into 2,271 Mbp with an N50 value of 10,563 bp. A genome sequence of 50.3 Mb was polished and assembled into 53 contigs with an N50 length of 1,785,794 bp, maximum contig length of 3,223,184 bp, and a sum of contig lengths of 51,182,778 bp. A total of 12,680 protein-coding genes were predicted using the Nonredundant, Gene Ontology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes, and SWISS-PROT databases. The genome sequence and annotation resources of R. oryzae provided a reference for studying its biological characteristics, trait-specific genes, pathogen-host interaction, pathogen evolution, and population genetic diversity. The phenomics and genome of R. oryzae will provide insights into microfungal biology, pathogen evolution, and the genetic diversity of epidemics.

Phyllospheric Microbial Composition and Diversity of the Tobacco Leaves Infected by Didymella segeticola.

A Myriad of biotic and abiotic factors inevitably affects the growth and production of tobacco (Nicotiana tabacum L.), which is a model crop and sought-after worldwide for its foliage. Among the various impacts the level of disease severity poses on plants, the influence on the dynamics of phyllospheric microbial diversity is of utmost importance. In China, recurring reports of a phyto-pathogen, Didymella segeticola, a causal agent of tobacco leaf spot, accentuate the need for its in-depth investigation. Here, a high-throughput sequencing technique, IonS5TMXL was employed to analyze tobacco leaves infected by D. segeticola at different disease severity levels, ranging from T1G (least disease index) to T4G (highest disease index), in an attempt to explore the composition and diversity of phyllospheric microbiota. In all healthy and diseased tobacco leaves, the most dominant fungal phylum was Ascomycota with a high prevalence of genus Didymella, followed by Boeremia, Meyerozyma and Alternaria, whereas in the case of bacterial phyla, Proteobacteria was prominent with Pseudomonas being a predominant genus, followed by Pantoea. The relative abundance of fungi, i.e., Didymella and Boeremia (Ascomycota) and bacteria, i.e., Pseudomonas and Pantoea (Proteobacteria) were higher in diseased groups compared to healthy groups. Healthy tissues exhibited relatively rich and diverse fungal communities in contrast with diseased groups. The infection of D. segeticola had a complex and significant effect on fungal as well as bacterial alpha diversity. FUNGuild analysis indicated that the relative abundance of pathotrophs and saprotrophs in diseased tissues proportionally increased with disease severity. PICRUSt analysis of diseased tissues indicated that the relative abundance of bacterial cell motility and membrane transport-related gene sequences elevated with an increase in disease severity from T1G to T3G and then tended to decrease at T4G. Conclusively, the current study shows the typical characteristics of the tobacco leaf microbiome and provides insights into the distinct microbiome shifts on tobacco leaves infected by D. segeticola.

First Report of Leaf spot Caused by Xylaria arbuscula on Flue-cured Tobacco in China.

Flue-cured tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves in China. Around 70% of tobacco production in China occurs in southwest China. In summer of 2019, leaf spot symptoms were observed on ten to twenty percent of tobacco plants in a 2 ha commercial field of Bijie (27.32° N, 105.29° E), Guizhou province, China. The leaf spots were white with dark-brown in edges, irregularly round and oval, and diseased tissue dropped out leaving the leaves ragged in appearance (Fig. 1A, 1B). One diseased leaf from each of five plants was sampled. From five leaves, a total of 15 small (5 mm × 5 mm) pieces of leaf tissue were cut from the edge of the lesions after surface sterilization and placed on potato dextrose agar (PDA) medium. Five fungal colonies that were similar in appearance were isolated and one was purified, BEZ22, was selected arbitrarily for identification. Mycelia of the pathogen was initally white and dense, and then black carbonized mycelia appeared from the center of the colony 7 days' after incubation. Mycelia was white, sparse and radiated when incubated on OA (oatmeal agar) (Fig. 1E, 1F, 1G, 1H). Genomic DNA of the isolate was extracted. The internal transcribed spacers (ITS) with primers ITS1/ITS4 (White et al. 1990), actin (ACT) gene with primers ACT-512F/ACT-738R (Hsieh et al. 2005), beta-tubulin (TUB2) with primers T1/T22 (O'Donnell & Cigelnik 1997) and RNA polymerase II second largest subunit gene (RPB2) with primers fRPB2-5F/ fRPB2-7cR (Liu et al. 1999) were amplified and sequenced, respectively. The generated sequences were deposited in GenBank with accession numbers MT804353 (ITS), MT809582 (ACT), MT799790 (TUB2) and MT799789 (RPB2). Using BLASTN searches, the sequences of each gene above were aligned with the voucher specimum, Xylaria arbuscula 89041211. The number of nucleotides that were similar for ITS (GU300090) was 550/551 (99%); for ACT (GQ421286), 266/266 bp (100%); for TUB2 (GQ478226), 1501/1501 bp (100%); and for RPB2 (GQ844805), 1135/1135 bp (100%), respectively (Fig. 2). A phylogenetic tree was constructed based on these four sequences with a final alignment of 3456 characters (ITS 551, ACT 266, TUB2 1501 and RPB2 1138). Thus, based on morphological and phylogenetic analyses, the isolate BEZ22 was identified as Xylaria arbuscula. To verify pathogenicity, six tobacco plants at seedling stage (5-6 leaves) without visible disease were inoculated using mycelial plugs (5 mm in diameter). Leaves inoculated with PDA only plugs served as controls. After inoculation, all tobacco plants were maintained in a greenhouse with 85% relative humidity at 25 oC under a 12/12 h light/dark cycle. Five days after inoculation, typical early symptoms were observed on the inoculated leaves, and not on the control leaves. Koch's postulates were fulfilled by re-isolation of the pathogen from diseased leaves. Xylaria arbuscula has also been reported as a pathogen of Macadamia in Hawaii (Wenhsiung et al. 2009) and sugarcane in Indonesia (Maryono et al. 2020). However, to our best knowledge, this is the first report of X. arbuscula causing leaf spot on tobacco in China. This leaf spot has the potential to cause serious damage to tobacco in this region that could result in reduced production, consequently disease management of this pathogen should be considered.

Fungal Composition and Diversity of the Tobacco Leaf Phyllosphere During Curing of Leaves.

Rhizopus oryzae causes tobacco pole rot in China during tobacco flue-curing. Flue-curing is a post-harvest process done to prepare tobacco leaves and involves three different stages: the yellowing stage has the lowest temperatures and highest humidity, then the color-fixing stage has higher temperatures and medium humidity, and finally the stem-drying stage has the highest temperatures and lowest humidity. In this study, fungal culturing and IonS5XL high-throughput sequencing techniques were used to reveal the fungal community of the petioles and lamina of tobacco leaves infected with pole rot during flue-curing. A total of 108 fungal isolates belonging to 6 genera were isolated on media. The most common fungal species isolated was the pathogen, R. oryzae, that was most often found equally on petioles and laminas in the color-fixing stage, followed by saprotrophs, mostly Aspergillus spp. High-throughput sequencing revealed saprotrophs with Alternaria being the most abundant genus, followed by Phoma, Cercospora, and Aspergillus, whereas Rhizopus was the tenth most abundant genus, which was mostly found on petioles at the yellowing stage. Both culturable fungal diversity and fungal sequence diversity was higher at stem-drying stage than the yellowing and color-fixing stages, and diversity was higher with leaf lamina than petioles revealing that the changes in fungal composition and diversity during the curing process were similar with both methods. This study demonstrates that the curing process affects the leaf microbiome of tobacco during the curing process, and future work could examine if any of these saprotrophic fungi detected during the curing of tobacco leaves may be potential biocontrol agents for with pole rot in curing chambers.

Metabolic Phenotype Characterization of Botrytis cinerea, the Causal Agent of Gray Mold.

Botrytis cinerea, which causes gray mold, is an important pathogen in four important economic crops, tomato, tobacco, cucumber and strawberry, in China and worldwide. Metabolic phenomics data on B. cinerea isolates from these four crops were characterized and compared for 950 phenotypes with a BIOLOG Phenotype MicroArray (PM). The results showed that the metabolic fingerprints of the four B. cinerea isolates were similar to each other with minimal differences. B. cinerea isolates all metabolized more than 17% of the tested carbon sources, 63% of the amino acid nitrogen substrates, 80% of the peptide nitrogen substrates, 93% of the phosphorus substrates, and 97% of the sulfur substrates. Carbon substrates of organic acids and carbohydrates, and nitrogen substrates of amino acids and peptides were the significant utilization patterns for B. cinerea. Each B. cinerea isolate contained 94 biosynthetic pathways. These isolates showed a large range of adaptabilities and were still able to metabolize substrates in the presence of the osmolytes, including up to 6% potassium chloride, 10% sodium chloride, 5% sodium sulfate, 6% sodium formate, 20% ethylene glycol, and 3% urea. These isolates all showed active metabolism in environments with pH values from 3.5 to 8.5 and exhibited decarboxylase activities. These characterizations provide a theoretical basis for the study of B. cinerea in biochemistry and metabolic phenomics and provide valuable clues to finding potential new ways to manage gray mold.