First Report of Alternaria alternata Causing White Leaf Spot on Allium tuberosum in China.

Puding County is the major Allium tuberosum growing area in Guizhou Province of China. In 2019, white leaf spots were observed on Allium tuberosum in Puding County (26.31°N, 105.64°E). The white spots, ranging from elliptic to irregular in shape, first appeared on leaf tips. With disease aggravation, spots gradually coalesced, forming necrotic patches with yellow margins causing leaf necrosis; sometimes there was gray mold on dead leaves. The incidence of the diseased leaf rate was estimated to be 27-48%. To identify the pathogenic agent, 150 leaf tissues (5 mm × 5 mm) were obtained from disease-healthy junctions of 50 diseased leaves. Leaf tissues were disinfected in 75% ethanol for 30 s, soaked in 0.5% sodium hypochlorite for 5 min, and flushed three times with sterile water, before being placed on potato dextrose agar (PDA) in the dark at 25 °C. When colonies appeared, the mycelial tips were picked and placed on new PDA. Purified fungus was obtained after repeating this last step several times. The colonies were grayish-green with white round margins. Conidiophores (2.7-4.5 µm × 27-81 µm) were brown, straight, or flexuous with branches and septa. Conidia (8-34 µm × 5-16 µm) were brown, with 0-5 transverse septa and 0-4 longitudinal septa. The 18S nuclear ribosomal DNA (nrDNA; SSU), 28S nrDNA (LSU), RNA polymerase II second largest subunit (RPB2), internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor 1-alpha (TEF-α) (Woudenberg et al. 2013) were amplified and sequenced. The sequences were deposited in GenBank (ITS: OP703616, LSU: OP860684, SSU: OP860685, GAPDH: OP902372, RPB2: OP902373, TEF1-α: OP902374). According to BLAST analysis, the ITS, LSU, GAPDH, RPB2, SSU, and TEF1-α of the straishowed 100% (689 of 731 base pairs; bp), 100% (916 of 938 bp), 100% (579 of 600 bp), 100% (946 of 985 bp), 100% (1093 of 1134 bp), and 100% (240 of 240 bp) sequence identity to those of Alternaria alternata (ITS: LC440581.1, LSU: KX609781.1, GAPDH: MT109295.1, RPB2: MK605900.1, SSU: ON055699.1 and TEF1-α: OM220081.1). A phylogenetic tree was constructed using PAUP4 and the maximum parsimony method with 1000 replicas of bootstrapping for all datasets. According to morphological characteristics and phylogenetic analysis, FJ-1 was identified as Alternaria alternata (Simmons 2007, Woudenberg et al. 2015). The strain was preserved in the Agricultural Culture Collection of China (preservation number: ACC39969). To determine the pathogenicity of Alternaria alternata against Allium tuberosum, wounded healthy leaves were inoculated with a conidial suspension (106 conidial/mL) and round mycelial plugs (4mm). Sterile agar PDA plugs with no mycelium or sterile water were inoculated as negative controls. Three days later, white spots appeared on the wounded leaves inoculated with mycelial plugs or conidial suspension. However, the symptoms caused by conidial suspensions were weaker than those caused by mycelial plugs. No symptoms were observed in the control group. The experimental symptoms were consistent with the phenomena observed in the field. The same fungus was reisolated from necrotic lesions and identified as Alternaria alternata using the method described above. To our knowledge, this is the first report of Alternaria alternata causing white leaf spots on Allium tuberosum in China, a disease seriously affected the yield and quality of Allium tuberosum and caused economic losses to farmers. Reference: Simmons EG (2007) Alternaria: an identification manual. CBS Fungal Biodiversity Centre, Utrecht, the Netherlands. Woudenberg JHC, Groenewald JZ, Binder M, Crous PW ( 2013) Alternaria redefined. Stud Mycol, 75: 171-212. https://doi.org/10.3114/sim0015. Woudenberg JHC, Seidl MF, Groenewald JZ, Vries M de, Stielow JB, Thomma BPHJ, Crous PW (2015) Alternaria section Alternaria: Species, formae speciales or pathotypes? Stud Mycol, 82:1-21. https://doi.org/10.1016/j.simyco.2015.07.001.

Draft Genome and Biological Characteristics of Fusarium solani and Fusarium oxysporum Causing Black Rot in Gastrodia elata.

Gastrodia elata is a valuable traditional Chinese medicinal plant. However, G. elata crops are affected by major diseases, such as brown rot. Previous studies have shown that brown rot is caused by Fusarium oxysporum and F. solani. To further understand the disease, we studied the biological and genome characteristics of these pathogenic fungi. Here, we found that the optimum growth temperature and pH of F. oxysporum (strain QK8) and F. solani (strain SX13) were 28 °C and pH 7, and 30 °C and pH 9, respectively. An indoor virulence test showed that oxime tebuconazole, tebuconazole, and tetramycin had significant bacteriostatic effects on the two Fusarium species. The genomes of QK8 and SX13 were assembled, and it was found that there was a certain gap in the size of the two fungi. The size of strain QK8 was 51,204,719 bp and that of strain SX13 was 55,171,989 bp. Afterwards, through phylogenetic analysis, it was found that strain QK8 was closely related to F. oxysporum, while strain SX13 was closely related to F. solani. Compared with the published whole-genome data for these two Fusarium strains, the genome information obtained here is more complete; the assembly and splicing reach the chromosome level. The biological characteristics and genomic information we provide here lay the foundation for further research on G. elata brown rot.

Fusarium solani Causing Root Rot Disease on Gastrodia elata in Shaxi, China.

Gastrodia elata, a traditional and important medicinal plant in China, it is used to numerous medical reasons. It is widely planted in Shaxi, Guizhou Province, China. G. elata grown in Guizhou is of high quality and an important source of income for the region. However, a root rot disease has been reported on G. elata in Guizhou in recent years, with an incidence rate of approximately 25%; this disease has markedly affected the plant growth and development. It causes what is referred to as a "rotten nest" and "empty nest", significantly reducing the yield and medicinal value of G. elata. Eighty diseased G. elata samples were collected from August to December 2020 in Shaxi. Tissue dissection was used to isolate the pathogen on an ultra-clean workbench. In short, thew surface of G. elata was wiped with 75% alcohol for 30 s and then rinsed three to four times with sterile water. After the surface had dried, the skin from an infected area of the plant was cut into a net shape using a sterile scalpel. Eighty diseased tissue samples were placed on PDA (potato dextrose agar) medium using a sterile medical syringe needle and placed in an incubator at 25 °C for 7 days, and 61 fungal isolates with the same morphological characteristics were obtained from the diseased samples. Pure cultures of a putative fungal pathogen designated SX13 were obtained using the single-spore isolation and cultured on PDA medioum for identification and analysis. The colony grew in a circular shape, and the early hyphae were compact and white. A light-yellow ring appeared in the outer circle of the hyphae, and could be seen on both sides of the plate. The upper side of the colony turned white subsequently, and the lower side was light yellow. Identification of SX13 as Fusarium solani was primarily done based on morphological characteristics (Chitrampalam et al., 2018). Colonies produced macroconidia, which were sickle-shaped with two to five septa; most of them had three septa (length by width: 17.28 to 36.23 µm by 4.33 to 6.43 µm). Smaller conidia were fusiform, renal, or oblong, with no or one septum (length by width: 5.56 to 14.35 µm by 2.93 to 5.76 µm). Chlamydospore were also observed with diameters of ranging from 3.43 to 13.12 µm. Identification of SX13 was verified through DNA sequencing. Genomic DNA was extracted using the Biomiga Fungal gDNA Kit. The internal transcribed spacer (ITS) region (primers ITS5/ITS4) (Schoch et al., 2012), ß-tubulin (primers T1/T2) (O'Donnell and Cigelnik, 1997), and actin gene (ACT) region (primers ACT-512F/ACT-783R) (Carbone and Kohn, 1999) were PCR amplified, sequenced, and subjected to NCBI BLASTn homology matching analyses (GenBank Accession Nos. MW888340, MW892976 and MZ440809). High levels of sequence homology were observed with a F. solani reference sequence (Accession Nos. MT560378, ITS=100%; KU938955, ß-tubulin=100%; KM231197, ACT=99%). To complete Koch's postulates, a conidial suspension (106 spores/mlcollected from isolate SX13 was inoculated onto nine G. elata root samples. Sterile water was used as a negative control, and the pathogenicity assay was repeated three times. Following inoculation, plants were kept under high relative humidity in the dark at 25 °C for 7 days. Symptoms similar to the original outbreak were observed on all inoculated plants. In contrast, the negative control plants were healthy and unaffected. The SX13 was re-isolated successfully from the diseased tissues and verified based on morphology and sequencing as described above. To the best of our knowledge, this is the first report of F. solani causing root rot disease on G. elata in China. These findings provide a basis for further research on the management of this disease.

The Chromosome-Scale Genomes of Exserohilum rostratum and Bipolaris zeicola Pathogenic Fungi Causing Rice Spikelet Rot Disease.

Rice spikelet rot disease occurs mainly in the late stages of rice growth. Pathogenicity and biological characteristics of the pathogenic fungus and the infestation site have been the primary focus of research on the disease. To learn more about the disease, we performed whole-genome sequencing of Exserohilum rostratum and Bipolaris zeicola for predicting potentially pathogenic genes. The fungus B. zeicola was only recently identified in rice.We obtained 16 and 15 scaffolds down to the chromosome level for E. rostratum LWI and B. zeicola LWII, respectively. The length of LWI strain was approximately 34.05 Mb, and the G + C content of the whole genome was 50.56%. The length of the LWII strain was approximately 32.21 Mb, and the G + C content of the whole genome was 50.66%. After the prediction and annotation of E. rostratum LWI and B. zeicola LWII, we predicted that the LWI strain and LWII strain contain 8 and 13 potential pathogenic genes, respectively, which may be related to rice infection. These results improve our understanding of the genomes of E. rostratum and B. zeicola and update the genomic databases of these two species. It benefits subsequent studies on the mechanisms of E. rostratum and B. zeicola interactions with rice and helps to develop efficient control measures against rice spikelet rot disease.