Abstract Ustilago maydis is a pathogenic fungus in Basidiomycota causing corn smut disease. A strain of U. maydis YZZF202006 was isolated from the tumor of corn smut collected from Jingzhou city in China. The intracellular bacteria were confirmed inner hyphal of the strain YZZF202006 by PCR amplification and fluorescence in situ hybridization (FISH) and SYTO-9. An endohyphal bacterium YZUMF202001 was isolated from the protoplasts of the strain YZZF202006. It was gram-negative, short rod-shaped with smooth light yellow colony. The endohyphal bacterium was genomic evidenced as Klebsiella michiganensis on the basis of average nucleotide identity (ANI) analysis and the phylogram. Then K. michiganensis was GFP-Labeled and reintroduced into U. maydis, which confirmed the bacterium can live in hyphae of U.maydis. The bacterium can grow on N-free culture media. Its nitrogenase activity was reached av. 646.25 ± 38.61 nmol·mL− 1·h− 1 C2H4 by acetylene reduction assay. A cluster of nitrogen fixation genes (nifJHDKTXENXUSVWZMFLABQ) was found from its genome. The endohyphal K. michiganensis may play an important role to help nitrogen fixation for fungi in the future.

Cymbidium sinense (Jackson ex Andr.) Willd is a perennial terrestrial plant in the orchid family mainly distributed in China, Japan, India and Southeast Asia that occupies a strong position in the flower market due to its bright green leaves and fragrant flowers (Zhang et al. 2013). Cymbidium sinense is not only valued by people for its ornamental and economic value, but its roots have antiasthmatic medicinal properties (Ke et al. 2004). In August 2020, about 15% stem rot on two-year old C. sinense with varying severity was observed in five nursery gardens located in Enshi city (N 30° 16', E 109° 29'), Hubei province, China. Typical symptoms of C. sinense included roots and inner part of the pseudobulbs changing from white to brown and rotting. Leaves became brown and withered from bottom to top, and there was an obvious blight yellow halo at the junction of diseased and healthy tissue, which eventually caused the whole plant to wilt and die (Fig. 1d). To isolate the pathogen, a total of 15 leaf tissues from the disease-health junction (3 × 3 mm) from 5 individual plants (3 leaves/plant) with symptoms were surface sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite (NaOCl) for 3 min. The sterilized tissue was rinsed three times with sterilized water, and then placed on potato dextrose agar (PDA) for incubation at 28°C in the dark for 5 days. Isolated colonies were subcultured by a hyphal tip protocol. Thirteen fungal isolates were obtained. Through preliminary pathogenicity tests, we found that ten isolates induced leaf blight. These ten isolates with pathogenicity showed similar morphological characteristics, with initial white-flocculent aerial mycelium that secreted a lavender pigment and produced colonies with an irregular edge after 3 days on PDA. The ten strains were cultured on PDA plates at 28℃ for 5 and 15 days to observe colony and conidial characteristics. The ten strains were identified as Fusarium based on morphological characteristics (Leslie and Summerell 2006). Strain ML0303 was selected for further identification. Macroconidia were falciform, hyaline, slightly pointed at both ends with two to four septa, 24.0 ± 5.6 µm × 4.7 ± 0.8 µm (n = 50). Microconidia were hyaline, oval, globose, with zero to one septum, 5.5 ± 1.3 µm × 2.2 ± 0.5 µm (n = 50) (Fig. 1c). Total genomic DNA of strain ML0303 was extracted with a CTAB protocol (Stenglein and Balatti 2006). The translation elongation factor (EF-1α), RNA polymerase II second largest subunit (RPB2) and β-tubulin (Tub2) genes were amplified respectively using primer pairs EF1/EF2, RPB2-5F2/RPB2-7cR and T1/T22 respectively (O'Donnell. et al. 2010, O'Donnell. et al. 1997). The EF-1α, RPB2 and Tub2 (accession numbers-MW719874, OL614838, OL689398, respectively) gene sequences were submitted to GenBank. EF-1α, RPB2 and Tub2 sequences of ML0303 showed 99.5% - 100% identity respectively with Fusarium oxysporum in the Genbank and FUSARIUM-ID databases. The multilocus sequence data was used to infer a phylogenetic tree via a Neighbor-joining (NJ), Maximum-likelihood (ML) and Maximum-Parsimony(MP) together with reference sequences from GenBank. The topology of the three trees was similar; only the NJ tree is presented here. Strain ML0303 and F. oxysporum formed a clade supported with high values (NJ/ML/MP: 96,95,97). The results indicated that the fungus was F. oxysporum based on the phylogenetic analysis and BLASTn queries. For pathogenicity tests, conidia of strain ML0303 were collected by rinsing PDA plates. Two-year-old C. sinense grown in plastic pots filled with sterilized autoclaved sandy loam soil were used for the tests. Three pots (two plants/pot) were included in each treatment. Spore suspensions (106spores/ml) of strain ML0303 were used to irrigate the stem-zone of the plants, and sterile water was used as control. The two treatments were placed in a greenhouse and incubated at 28±2℃ with a 14-hour light/10-hour dark cycle. The experiment was repeated twice. After three weeks, stem rot symptoms were observed on C. sinense inoculated with ML0303, that were the as same as observed in the nursery (Fig. 1e-h). No symptoms were observed on the negative control. Fusarium oxysporum was re-isolated from the infected plants to fulfill Koch's postulates. Partial EF-1α and RPB2 gene sequences were used for molecular identification. Members of the FOSC are notorious for causing many diseases, which includes stem rot of Sulcorebutia heliosa and root rot of Torreya grandis (Garibaldi et al. 2020; Zhang et al. 2016). To our knowledge, this is the first report of stem rot by F. oxysporum on C. sinense in China. The finding of this pathogen provides a clear target for stem rot control.

suspension (10 6 conidia/mL) by the root-drenching.Control plant roots were immersed in water.After three weeks, symptoms like the original ones were observed only on inoculated plants.The identity of the fungus was confirmed after re-isolation.To our knowledge, this is the first report of F. odoratissimum (FOSC) causing root rot on P. hadiensis in China.

Fusarium oxysporum KB-3 had been reported as a mycorrhizal fungus of Bletilla striata, which can promote the seed germination and vegetative growth. Endohyphal bacteria were demonstrated in the hyphae of the KB-3 by 16S rDNA PCR amplification and SYTO-9 fluorescent nucleic acid staining. A strain Klebsiella aerogenes KE-1 was isolated and identified based on the multilocus sequence analysis. The endohyphal bacterium was successfully removed from the wild strain KB-3 (KB-3−), and GFP-labeled KE-1 was also transferred to the cured strain KB-3− (KB-3+). The production of indole-3-acetic acid (IAA) in the culturing broths of strains of KE-1, KB-3, KB-3−, and KB-3+ was examined by HPLC. Their IAA productions were estimated using Salkowski colorimetric technique. The highest concentrations of IAA were 76.9 (at 48 h after inoculation), 31.4, 9.6, and 19.4 μg/ml (at 60 h after inoculation), respectively. Similarly, the three fungal cultural broths exhibited plant promoting abilities on the tomato root and stem growth. The results indicated that the ability of mycorrhizal Fusarium strain KB-3 to promote plant growth was enhanced because its endohyphal bacterium, Klebsiella aerogenes KE-1, produced a certain amount of IAA.

Raspberry (Rubus rosaefolius Smith), also called march bubble or milk bubble, is widely distributed and economically important in China. Raspberries are rich in nutrients such as essential amino acids, vitamin C, dietary fiber, superoxide dismutase (SOD) and minerals (Yang et al. 2019). In May 2019, a leaf spot disease was observed on raspberry in Enshi (N29°07'10', E108°23'12'), Hubei province of China. The symptoms were small dark-brown spots (Fig.1) on over 90% of observed plants. To isolate the pathogen, leaf sections (5 mm × 3 mm) from the border of the symptomatic tissue were cut and sterilized with 75% ethanol for 30 s, followed by 2% sodium hypochlorite (NaClO) for 2 min, and then rinsed three times with sterile water. Leaf sections were placed on potato dextrose agar (PDA) medium amended with 25 μg / ml ampicillin and incubated at 25 °C in the dark for 3 days. Isolated colonies were sub-cultured on PDA by hyphal tip transfer. Eight fungal isolates with similar morphology, abundant white aerial hyphae, were collected. Colonies on PDA grew up to 80 mm in diameter by 7 days at 25 °C. The center of each colony became black (Fig.2). Conidia were unicellular, oval and hyaline. Conidia ranged in size from 14.5 to 19.75 µm × 5.80 to 10.20 µm (n=50) in 20% (v/v) V8 vegetable juice medium. No appressoria were observed. Morphological characteristics are similar to those of Colletotrichum spp. (Moriwaki et al. 2003). Total genomic DNA of a representative isolate S1 was extracted with a CTAB method (Stenglein et al. 2006). Internal transcribed spacer (ITS) region of rDNA, actin (ACT) , beta-tubulin (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced with the primer pairs of ITS4 / ITS5, ACT512F / ACT783R, Bt-2a / Bt-2b and GDF1 / GDR1, respectively (Weir et al. 2012). BLAST results showed that ITS, ACT, TUB2 and GAPDH gene sequences (GenBank accession nos. MN498030, MT780498, MT780496 and MT780497, respectively) were 99% identical to those of Colletotrichum boninense Moriwaki, Sato & Tsukiboshi (GenBank accession nos. MF076598, JX009583, JQ005588 and JX009905, respectively). Concatenated sequences of the four genes were used to conduct a phylogenetic analysis using neighbor-joining method in MEGA7 (Toussaint et al. 2016). The isolate S1 clustered with above C. boninense strains retrieved from NCBI database. Therefore, the present isolate S1 was identified as C. boninense. Pathogenicity tests were performed using one-month-old raspberry plants, 24 controls and 30 inoculated. The plants were sprayed with conidial suspension ( 106 conidia / mL) cultured on 20% (v/v) V8 vegetable juice medium for 15 days. The control plants were sprayed with sterile distilled water. All plants were covered with plastic bags 24h to maintain the relative humidity in the field. Fifteen days after inoculation, typical symptoms of brown spots were observed on leaves similar to the disease on field plants, while the leaves from the control group remained asymptomatic. C. boninense was reisolated and identified from inoculated symptomatic leaves. Anthracnose on raspberry caused by Colletotrichum gloeosporioides (Dai et al. 2013) and C. fioriniae (Schoeneberg et al. 2020) has previously been reported. However, to the best of our knowledge, this is the first report of Colletotrichum boninense causing leaf spot on Raspberry in China. If more reports of this pathogen are found on raspberries, then it may be necessary to develop effective management strategies for controlling this disease.

Akebia trifoliata (Thunb.) Koidz. is a species in the family Lardizabalaceae, which belongs to deciduous woody lianas. It is an important species of plant used in Chinese medicine. In July 2019, a leaf spot disease was observed on A. trifoliata in a nursery garden in Jingzhou (N 30° 21', E 112° 19'), Hubei Province, China. Symptoms initially appeared as small brown spots and subsequently developed into subcircular or irregular-shaped brown necrotic lesions. In severe cases, the leaves became completely necrotic and abscised. The incidence of leaf symptoms on affected plants ranged was between 30% and 40%. To isolate the pathogen, pieces of symptomatic leaves were collected and excised at the margins of lesions, surface disinfected with 70% ethanol and 0.1% HgCl2, rinsed three times with sterile water, placed on potato dextrose agar (PDA) amended with 50 μg/ml kanamycin, and incubated at 28°C in the dark for 3 days. Isolated colonies were subcultured by transferring hyphal tips. Six fungal isolates were isolated from the collected tissues. All six isolates had similar colony morphologies on on PDA and were composed of white flocculent aerial hyphae. The average radial growth rate of colonies after 7 days was 11.2 mm/d. Isolates were later cultured on 20% V8 juice agar for 20 days to encourage sporulation. Sporangia were produced on V8 media and were colorless, inverted, pear-shaped, and terminal, with obvious mastoid, 22 to 34 × 28 to 46µm (n=50); Oospores were light brown, and suborbicular, with thick wall, 18 to 26µm (n=20); Globose chlamydospores were light brown, and suborbicular, 12 to 32µm (n=50). Antheridia were not observed suggesting homothallism. These morphological charactertistics were identical to those reported for Phytophthora nicotianae (Erwin and Ribeiro 1996). We selected a single isolate 'B2', for molecular identification because it was the most aggressive in leaf pathogenicity assays. The internal transcribed spacer (ITS) region of rDNA was amplified and sequenced using primers ITS1/ITS4 (White et al. 1990). BLAST analyis revealed that the ITS sequence (GenBank accession nos. MT472132) was 100% identical to other P. nicotianae strains (GenBank accession nos. KJ754387). To fulfill Koch's postulates, a 50 ml zoospores suspension (106 spores/ml) of B2 was sprayed on the foliage of three 1-year-old healthy seedlings. Sterile distilled water to inoculate control plants. After 10 days, typical symptoms of dark brown spots were observed on all the inoculated leaves, while the control leaves remained asymptomatic. P. nicotianae was re-isolated from the inoculated, symptomatic leaves, thus confirming Koch's hypothesis. The experiment was repeated three times. To the best of our knowledge, this is the first report of P. nicotianae causing leaf spot on A. trifoliata in China. P. nicotianae is a common stramenopile pathogen that infects many plant hosts. The presence of this pathogen in an A. trifoliata nursery should be carefully considered to mitigate possible outbreaks of this disease in other fields in this growing region.

HomePlant DiseaseVol. 104, No. 2First Report of Damping-Off in Actinidia argute (Tara Vine) Caused by Fusarium proliferatum in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Damping-Off in Actinidia argute (Tara Vine) Caused by Fusarium proliferatum in ChinaJian-wei Jiang, Peng-yu Liang, Sheng Cheng, Qian-wen Nie, Jun-ping Liu, Tom Hsiang, Zheng-xiang Sun, and Yi ZhouJian-wei JiangCollege of Agriculture, Yangtze University, Jingzhou, Hubei, 434025, ChinaSearch for more papers by this author, Peng-yu LiangCollege of Agriculture, Yangtze University, Jingzhou, Hubei, 434025, ChinaSearch for more papers by this author, Sheng ChengCollege of Agriculture, Yangtze University, Jingzhou, Hubei, 434025, ChinaSearch for more papers by this author, Qian-wen NieCollege of Agriculture, Yangtze University, Jingzhou, Hubei, 434025, ChinaSearch for more papers by this author, Jun-ping LiuJingzhou Institute of Technology, Jingzhou, Hubei, 434025, ChinaSearch for more papers by this author, Tom HsiangEnvironmental Sciences, University of Guelph, Guelph, Ontario, CanadaSearch for more papers by this author, Zheng-xiang Sun†Corresponding authors: Z. Sun; E-mail Address: sunzhengxiang9904@126.com and Y. Zhou; E-mail Address: zhouyi@yangtzeu.edu.cnhttp://orcid.org/0000-0001-5009-5384College of Agriculture, Yangtze University, Jingzhou, Hubei, 434025, ChinaSearch for more papers by this author, and Yi Zhou†Corresponding authors: Z. Sun; E-mail Address: sunzhengxiang9904@126.com and Y. Zhou; E-mail Address: zhouyi@yangtzeu.edu.cnCollege of Agriculture, Yangtze University, Jingzhou, Hubei, 434025, ChinaSearch for more papers by this author AffiliationsAuthors and Affiliations Jian-wei Jiang1 Peng-yu Liang1 Sheng Cheng1 Qian-wen Nie1 Jun-ping Liu2 Tom Hsiang3 Zheng-xiang Sun1 † Yi Zhou1 † 1College of Agriculture, Yangtze University, Jingzhou, Hubei, 434025, China 2Jingzhou Institute of Technology, Jingzhou, Hubei, 434025, China 3Environmental Sciences, University of Guelph, Guelph, Ontario, Canada Published Online:4 Dec 2019https://doi.org/10.1094/PDIS-07-19-1537-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Actinidia argute (tara vine) is a deciduous, fast-growing, twining woody vine typically grown for its attractive foliage and edible fruit and native to Japan, Korea, northern China, and the Russian Far East (Latocha et al. 2013). In March 2019, severe damping-off was observed in a base for seedling cultivation of A. argute located on Taihu farm of Jingzhou (N 111°15′, W 29°26′), Hubei province, China. Disease incidence in variety Longcheng No. 2 was approximately 30%, which resulted in severe seedling loss. At the early stage of the disease, seedlings showed symptoms of light to dark brown lesions on the lower stems near the soil and root necrosis. Finally, the seedlings became wilted and rotted. To isolate the pathogen, stem sections (3 × 2 mm) from 20 individual plants with symptoms were surface sterilized with 75% ethanol for 30 s and 2% NaOCl for 1 min. The sterilized tissue was rinsed four times with sterilized water and then put on potato dextrose agar (PDA) amended with 50 μg/ml of ampicillin and kanamycin to incubate at 25°C in the dark for 5 days. Isolated colonies were subcultured by the hyphal tip transferring method. Sixteen fungal isolates were obtained from 16 sterilized tissues, except for four tissues. Through the preliminary inoculation tests, we found that 13 isolates showed damping-off symptoms, except for three isolates. These 13 isolates with pathogenicity showed similar morphological characteristics with white to salmon pink colonies on PDA and abundant white aerial hyphae. Mung bean soup (MBS) medium was used to stimulate conidia production. Macroconidia were long, sickle shaped, three to four septa, 37.29 ± 6.03 × 3.56 ± 0.40 μm (n = 50), and microconidia were short oval, zero to one septa, 8.92 ± 1.74 × 2.88 ± 0.36 μm (n = 50). The isolation was identified as Fusarium sp. based on morphological characteristics (Leslie and Summerell 2006). Total genomic DNA of 13 isolates was extracted with the CTAB method (Stenglein and Balatti 2006). The ITS gene of all were sequenced using primers ITS4/ITS5 (White et al. 1990) and differed by only 1 or 2 bp of 466 nt total. A representative isolate, RZ1901, was used for further characterization using translation elongation factor 1α (EF-1α) (EF-728F/EF-986R) (Carbone and Kohn 1999) and β-tubulin genes (BT2-a/BT2-b) (Glass and Donaldson 1995), respectively. ITS (accession no. MN095191), EF-1α (MN227186), and β-tubulin (MN103540) sequences were deposited in GenBank. Three sequences were analyzed via FUSARIUM-ID (http://fusariumdb.org/index.php) and Fusarium MLST (http://www.wi.knaw.nl/fusarium/) and showed at least 99.57% identity to F. proliferatum (CBS130179, CBS 217.76, and NRRL 22109). For the pathogenicity test, isolate RZ1901 was cultured in MBS for 1 week to get spore suspension. Stems and main roots of 20 healthy 2.5-month-old A. argute seedlings were wounded by sterilized insect needle and soaked in RZ1901 conidial suspension (106 spores/ml) for 60 min, and 10 seedlings with sterile distilled water were used as a control. All plants were transplanted to pots filled with autoclaved (121°C) peat moss and placed in a greenhouse (25 ± 2°C, 65% relative humidity). After 3 weeks, damping-off symptoms were observed on inoculated seedlings, similar to the diseased seedlings in the field. But there were no symptoms for the controls. The experiment was repeated twice. Reisolation was performed with surface-sterilized tissue from symptomatic seedlings in one experiment. Fungal isolates were confirmed as F. proliferatum based on morphological and molecular characters. F. proliferatum, a soilborne pathogen with a wide host range, has been reported to cause soybean damping-off in the United States (Díaz Arias et al. 2011). To our knowledge, this is the first report of damping-off by F. proliferatum on A. argute in China. The intensity and crop loss of this disease will be carefully monitored to determine if management strategies are justified for control.The author(s) declare no conflict of interest.References:Carbone, I., and Kohn, L. M. 1999. Mycologia 91:553. https://doi.org/10.1080/00275514.1999.12061051 Crossref, ISI, Google ScholarDíaz Arias, M. M., et al. 2011. Plant Dis. 95:1316. https://doi.org/10.1094/PDIS-04-11-0346 Link, ISI, Google ScholarGlass, N. L., and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61:1323. Crossref, ISI, Google ScholarLatocha, P., et al. 2013. J. Sci. Food Agric. 93:1412. https://doi.org/10.1002/jsfa.5909 Crossref, ISI, Google ScholarLeslie, J. F., and Summerell, B. A. 2006. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, U.K. doi.org/10.1002/9780470278376 Crossref, Google ScholarStenglein, S. A., and Balatti, P. A. 2006. Physiol. Mol. Plant Pathol. 68:158. https://doi.org/10.1016/j.pmpp.2006.10.001 Crossref, ISI, Google ScholarWhite, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Crossref, Google ScholarThe author(s) declare no conflict of interest.Z. Sun and Y. Zhou contributed equally to the paper.Funding: This work was supported by National Key Research and Development Program (2017YFD0201106) and Excellent Doctor’s and Master’s Degree Thesis Cultivation Program of Yangtze University (YS2018033).DetailsFiguresLiterature CitedRelated Vol. 104, No. 2 February 2020SubscribeISSN:0191-2917e-ISSN:1943-7692 DownloadCaptionSymptom of maize ear rot caused by Fusarium sporotrichioides (B. B. Wang et al.). Photo credit: C. X. Duan. Systemic symptoms of alfalfa mosaic virus (AMV) isolate CaM on leaves of potato (X. Z. Nie et al.). Photo credit: X. Z. Nie. Metrics Downloaded 2,295 times Article History Issue Date: 31 Jan 2020Published: 4 Dec 2019First Look: 7 Oct 2019Accepted: 2 Oct 2019 Page: 578 Information© 2020 The American Phytopathological SocietyKeywordsfirst reportdamping-offActinidia arguteFusarium proliferatumThe author(s) declare no conflict of interest.