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.

First Report of Target Spot Caused by Rhizoctonia solani AG-6 in Tobacco in China.

Tobacco is an annual and solanaceous crop, which is widely produced in China. In July 2020, tobacco target spot was observed on 50% of tobacco plants in a 5-ha commercial field of Bijie (27.32° N, 105.29° E), Guizhou province, China. Typical symptoms firstly appeared on the old leaves as round watery spots. Then the spots became a diameter of 2 to 20 cm, with concentric ring lines and dead spots. Fifteen small pieces (5 × 5 mm) of leaf tissue were cut from the edge of the lesions, surface sterilized and placed on potato dextrose agar (PDA) medium amended with kanamycin (0.1 mg/ml). Isolate J136, one of five isolates with similar morphology, was selected for pathogen identification. The culture of the isolate on PDA was brown and exhibited radial mycelial growth after incubation at 28 oC in darkness for 5 days. Hyphae of the fungus were white at the beginning, turned light brown to brown at the later stages, and finally became thick and separated. Sclerotia were brown and produced on PDA after 25 days of incubation in the dark. These characteristics were similar to the colony characteristics of R. solani. The genomic DNA of Isolate J136 was extracted using the CTAB method. PCR analyses were conducted using the following primers specifically designed for the detection of individual AGs or subgroups of R. solani: AG-1 IA, IB and IC (Kuninaga 2003), AG-2-1, AG-2-2, IIIB, IV and LP (Carling et al. 2002), AG-3 PT (Misawa 2015), AG-4 HG-I and HG-II (Kuninaga 2003), and AGs-5-6 (Arakawa and Inagaki 2014). Among the 12 specific primer pairs, only AG-6-specific primers amplified a fragment of ca. 230 bp product, indicating that the tested strain belonged to R. solani AG-6. The sequence was deposited in GenBank with accession no. MZ379468. Using BLASTN search, the sequence of the gene was aligned with the voucher specimen, R. solani AG-6. A phylogenetic tree was constructed based on these sequences. After wards, Isolate J136 was tested for hyphal anastomosis reaction using the R. solani AG-6 standard strain according to the method described by Ogoshi (1987). The hyphal diameter at the point of anastomosis was reduced, with obvious anastomosis point, and the death of adjacent cells, indicating their anastomosis reactions (Anderson 1982). Thus, based on the morphological and genetic analyses, the fungus was identified as R. solani AG-6. To verify its pathogenicity, six plants (cv. Yunyan87) at the 5-to-6 leaf stage were inoculated with mycelial PDA plugs (5 mm in diameter). Leaves inoculated with PDA-only plugs served as the controls. Treated tobacco plants were maintained at a temperature range of 15 to 25 oC in a greenhouse with 85% relative humidity. After 5 days inoculation, typical symptoms were observed on the inoculated leaves, whereas no symptoms were observed on the control leaves. Koch's postulates were fulfilled by re-isolation of the pathogen from the diseased leaves. R. solani AG-2-2 is the only previously reported group of R. solani, which causes tobacco target spot in the field (Gonzalez et al. 2011). Therefore, to our knowledge, this is the first report of R. solani AG-6 causing target spot of tobacco in the field in China. Since considerable losses caused by the disease have frequently happened in this region, addition of this new group pathogen in the disease pool can be more problematic. Proper disease control strategies are in need to be developed to prevent further losses.

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.

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.

The Effects of Temperature and Humidity on a Field Population of Bradysia odoriphaga (Diptera: Sciaridae).

The production of Chinese chives is reduced throughout China due to a root-feeding dipteran pest Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae), therefore deciphering the conditions influencing its growth and development are important in developing ecological control strategies. A study was conducted from 2014 to 2017 to determine the relationship between the abundance of B. odoriphaga and temperature (atmospheric and soil), soil water content, and atmospheric humidity in a Chinese chive field in Beijing City, China. Numbers of adults peaked in March and October to November and were lowest in July to August and December to next February; numbers of larvae were highest in December to next February and lowest in July to August. From 2014 to 2017, the numbers of adults and larvae were significantly correlated with monthly mean atmospheric temperatures and soil temperatures, but were not significantly correlated with monthly mean atmospheric relative humidity and soil water content. However, for both adults and larvae, numbers were significantly greater with high soil water contents compared with drought treatment. The results of this study suggest that the very low soil water contents, high atmospheric temperatures, and high soil temperatures were critical for regulating field populations of B. odoriphaga.

Effect of Sex and Air Temperature on the Flight Capacity of Bradysia odoriphaga (Diptera: Sciaridae).

Bradysia odoriphaga Yang & Zhang (Diptera: Sciaridae) is an important pest of Chinese chives. Information on the effects of biotic and abiotic factors on the flight performance of B. odoriphaga is crucial for understanding the pest's ability to disperse and migrate. In this study, the effects of sex and air temperature on the flight performance of B. odoriphaga imagoes were assessed by tethering individual imagoes to computerized flight mills for a 10-h experiment. The results showed that the percentage of imagoes that flew a particular distance gradually decreased as flight distance increased. The percentage of imagoes was significantly higher for males than females when the flight distance was <300 m. Sex and air temperature significantly affected average flight time (which ranged from 14.6 to 68.3 min) and average flight distance (which ranged from 10.4 to 107.2 m), but did not significantly affect average flight speed (which ranged from 3.8 to 6.4 m/min). For both females and males, the average flight distance and flight time were shortest at 18°C and longest at 22°C; the interaction between air temperature and sex was not significant. The results suggest that B. odoriphaga has a poor potential for long-distance migration. These findings will be helpful for developing forecasting and management systems for B. odoriphaga.

Control of Bradysia odoriphaga (Diptera: Sciaridae) With Allyl Isothiocyanate Under Field and Greenhouse Conditions.

The botanical compound allyl isothiocyanate (AITC) is toxic to many microorganisms and insects. The aim of this study was to assess the effects of AITC on the Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae) and the seeds and seedlings of the Chinese chive. Allyl isothiocyanate was toxic to all four developmental stages of B. odoriphaga. The adult was significantly more sensitive to AITC than the other three stages, which exhibited no significant differences to one another in sensitivity to the chemical. The control efficacy of AITC against B. odoriphaga was far superior in the greenhouse than the field. In addition, seedling survival was higher in the greenhouse compared with that in the field. In the absence of B. odoriphaga, seed germination and seedling growth of Chinese chives were inhibited by 16 µl/liter of AITC, and significant inhibition occurred under higher doses of AITC. These results indicate that AITC could be used to control B. odoriphaga during cultivation of Chinese chives.