Jujube (Zizyphus jujuba Mill.), a native small deciduous tree of China, is widely cultivated in China, Korea, India, Japan, Europe, and the United States (Chen et al. 2020). The fruit have been commonly consumed as healthy food supplements and traditional Chinese medicine for over 2000 years (Li et al. 2007). In August 2019, anthracnose-like leaf spot symptoms were observed on jujube plants in Xiaomenya Village, Jinan City, Shandong Province, China (36°27'39″N, 117°3'13″E), with over 30% leaf disease incidence. The spots were circular, sunken, brown in the center and with dark brown edges. As the spots enlarged and coalesced, it resulted in leaf perforation and early defoliation. Sometimes acervuli were observed on the lesions (Fig. S1a, b). To identify the causal agent, 20 diseased leaves were sampled, the margins of the lesions were cut into pieces (5 × 5 mm), sterilized and cultured following the protocol described previously (Wan et al. 2020) at 25 ℃ for 5 days. Twelve monospore isolates showing identical colony morphology were obtained. Three representative isolates, JNZG11, JNZG311, JNZG313, were used for further study. When grown on PDA the colony color was initially white and then turned pale-gray to gray in 5-day-old cultures. On the reverse, colonies were brown-black with an orange pigmentation near the center. Aerial mycelium was cottony, dense, white to pale-gray. Conidia were hyaline, 1-celled, smooth-walled, subcylindrical, oblong, attenuated with slightly rounded ends, (11.1-) 12.7-13.3 (-17.8) ×(-4.4) 5.2-5.5 (-6.3) μm (n=50). Appressoria were dark-brown, oval or irregular, (7.3-) 8.6-9.2 (-9.8) ×(-5.1) 5.8-6.9 (-7.0) μm (n=50) (Fig. S1c-g). The morphology resembled those of Colletotrichum gloeosporioides species complex (Cannon et al. 2012). For accurate identification, the sequences of the ribosomal internal transcribed spacer (ITS), actin (ACT), β-tub2 (TUB2), calmodulin (CAL), chitin synthase (CHS-1), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) of the 3 isolates were sequenced (Weir et al. 2012), and deposited into GenBank (Accession Nos. see Table 1). The six loci (ITS, GAPDH, ACT, CHS-1, CAL, and TUB2) were concatenated and the aligned sequences (1904 bp) were 99.7% homologous to ex-type C. siamense ICMP18578. The sequences of 38 Colletotrichum species (44 isolates) were downloaded from GenBank for phylogenetic analyses. In the maximum likelihood phylogenetic tree generated, the highest log likelihood was -8798.90 and the three isolates were all in the C. siamense clade (bootstrap support 94 %) (Fig. S2). To complete Koch's postulates, 60 healthy, mature jujube leaves on 12 branches (5 leaves per branch) (variety 'Zhongqiuhong') were inoculated with 20 μL of spore suspension (106 conidia/mL) or sterile water as a control. The branches were placed in sterile beakers containing a small amount of sterile water sealed with plastic wrap and maintained at 28 °C, 12 h light/dark. Five days after inoculation, all treated leaves showed the typical anthracnose symptom, similar to that observed in the field (Fig. S1h). The same fungus was re-isolated from the margins of the lesions using the aforementioned methods. Whereas no fungus were isolated from the controls. Previously, C. siamense has been reported to infect Z. mauritiana in China (Shu et al. 2020). To our knowledge, this is the first report of C. siamense causing anthracnose on Z. jujuba in China. This finding provides crucial information for the effective management of this disease.

Schima superba Gardn. et Champ. is a subtropical evergreen tree species naturally distributed mainly in China, Japan, and Vietnam. It is primarily planted for its timber and urban landscaping in China (Ni, 1996). In September 2018, leaves necrotic spots were observed on S. superba in Jiangxi Forest Breeding Center (28°57'19.52" N, 115°39'21.32" E), Jiangxi Province, China. The disease incidence was about 30%. Initially, spots were circular to semicircular, grayish-brown in the center with dark brown margin, then expanded and eventually collapsed into sunken necrotic lesions. To identify the agent, diseased leaves were collected randomly. Pieces (5 × 5 mm) from the lesion borders were surfaced sterilized in 70% ethanol (30 s), 3% NaOCl (60 s), and rinsed 3 times in sterile water. These pieces were put on potato dextrose agar (PDA) and cultured at 25 °C. Pure cultures were obtained by monosporic isolation, and 3 isolates (MH-1, MH-2, MH-3) were used for morphological studies and phylogenetic analyses. On PDA, colonies were initially white, cottony, then became pinkish to deep-pink at the center and pink on the reverse. Conidia were fusiform with acute ends, smooth-walled, hyaline, 13.7-18.5 × 4.6-6.1 µm (16.4 ± 1.3× 5.3 ± 0.6 µm, n = 100). Conidiophores were colorless to pale brown, smooth, septate. Conidiogenous cells were colorless to pale brown, smooth, cylindrical to ampulliform. The morphological characteristics fit the descriptions of Colletotrichum acutatum J. H. Simmonds sensu lato (Damm et al., 2012). For accurate identification, genomic DNA of 3 isolates was extracted, and the internal transcribed spacer (ITS), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin 2 (TUB2), and chitin synthase (CHS-1) were amplified and sequenced using the corresponding primers (Weir et al., 2012). The sequences were deposited in GenBank (ITS: MZ325946, MZ325947, MW584318; ACT: MZ399375, MZ419566, MW661171; CHS-1: MZ399376, MZ419567, MW661172; MZ399377, GAPDH: MZ419568, MW661173; TUB2: MZ399378, MZ419569, MW661174). Five loci were concatenated, and the aligned sequences (1528bp) were 99.89% homologous to ex-type C. fioriniae (Marcelino & Gouli) R. G. Shivas & Y. P. Tan CBS128517. Phylogenetic analysis using the maximum likelihood showed that 3 isolates were clustered in C. fioriniae clade with 100% bootstrap support. Based on the multi-locus phylogeny and morphology, 3 isolates were identified as C. fioriniae. Pathogenicity tests were performed on 36 seedlings of S. superba (2-year-old). The leaves were wounded slightly and inoculated with a drop of spore suspension (106 conidia/mL). The sterile water was used as controls. All the tested leaves were covered with black plastic bags to keep them moist for 2 days. All seedlings were placed in the greenhouse (25 °C, 12 h light/dark) for 10 days, and all inoculated leaves had typical symptoms. The controls were asymptomatic. The same fungus was reisolated from the lesions, fulfilling Koch's postulates. Colletotrichum fioriniae was described as a new species from the C. acutatum s. l. (Shivas et al., 2009), and it was an important plant pathogen, such as Pyrus spp. (Pavlović et al., 2019), Morus alba L. (Xue et al., 2019), and so on. This is the first report of the newly emerging disease of S. superba caused by C. fioriniae in the world, and its potential threat should be evaluated in the future. This study provided crucial information for epidemiologic studies and appropriate control strategies.

As one of the major forest diseases in the world today, Bursaphelenchus xylophilus is difficult to diagnose and treat, causing huge economic losses. At present, preliminary studies have been made on the early diagnosis of Bursaphelenchus xylophilus through changes in the structure of the microbial flora. However, according to the complex wild growing environment, there are still no related scholars to study and report the differences in the microbial flora structure with different degrees of disease and different sampling locations. In the experiment of this study, by analyzing bacteria in different ecological tissue parts of different disease levels of Pinus massoniana in the field with high-throughput sequencing technology, it was found that the structures of the in vivo flora of healthy Pinus massoniana and different parts of the diseased pine affected by pine wood nematode were significantly different. The results showed that the bacteria in healthy Pinus massoniana was mainly composed of unidentified Oxyphotobacteria, Sphingomonas leidyi, Beijerinckia sp M016010, Methylobacterium komagatae, and unknown bacteria. After inoculation with pine wood nematodes, the bacterial flora diversity in the Pinus massoniana samples increased with the increase of the infection time and disease level. Along with the sharp decrease in unidentified Oxyphotobacteria, the bacteria abundance of Sphingomonas leidyi, Paradoxia multiseta, Luteibacter rhizovicinus, Burkholderia sp symbiont of Dicranocephalus medius, Elliptochloris bilobata, Beijerinckia sp M016010, Clostridium disporicum, Methylobacterium komagatae and Amantichitinumursilacus all increased. With horizontal comparison of three different tissue niches of Pinus massoniana, it was found that during the pinewood nematode infestation, the flora structures of pine needles and pine branches were similar, showing a sharp decrease in the abundance of unidentified Oxyphotobacteria, but a significant increase in the abundance of Paradoxia multiseta, Burkholderia sp symbiont of Dicranocephalus medius, Beijerinckia sp M016010, and unknown bacteria. However, the structural diversity of pine stems was slightly different, which was mainly manifested by a significant increase in the abundance of Clostridium disporicum, Luteibacter rhizovicinus, and unknown bacteria. At the same time, after different tissue parts were infected by pine wood nematode, the flora structure of Pinus massoniana changed significantly when the first-level disease characterization occurred. From the diversity and richness of the detected flora structure, it can be seen that the bacterial flora structures of pine branches and pine needlesare similar, and their bacteria abundancesare higher than that of pine stems, and the abundance of the pine branches is the highest. By analyzing the differences in the bacterial flora structure in the three major ecological nichesofdiseased Pinus massoniana with different levels of Bursaphelenchus xylophilus, this study provides data support for the further use of the differences in microbial flora structure to diagnose Bursaphelenchus xylophilus in Pinus massoniana. Meanwhile, it provides technical guidance at the selection of sampling and detecting sites.

With the environmental and ecological security problems caused by the continuous use of chemical fertilizers and pesticides, more and more biological agents have been developed and used in recent years. Through in-depth analysis of the effects of two kinds of biological agents on microbial diversity in the rhizosphere of pepper seedlings, this study exploresprobiotic strains that promote growth and disease resistance, and provides some theoretical basis and data support for farmers to purchase and select biological agents. In the experiment of this study, two different kinds of biological agent were applied to the roots of pepper seedlings, whose rhizosphere soil was collected after 10 days, 20 days, 30 days and 40 days respectively. The total DNA, of bacteria in rhizosphere soil was extracted by soil genomic DNA extraction kit with magnetic bead method. Besides, the experiment used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique to analyze the effects of two biological agents on bacterial diversity in the rhizosphere soil of pepper seedlings. The results showed that the total abundance and species of microflora in the rhizosphere soil of pepper seedlings irrigated with Bacillus subtilis and Acinetobacter sp. increased, while the plant growth in the Bacillus subtilis treatment group and Acinetobacter sp. treatment group was significantly better than that of the blank control group, and the Bacillus subtilis treatment group was the best. It was further found that Uncultured bacterium clone (JF2360 35), Bacillus firmus OrfA gene (U61539), Uncultured Gemmatimonadete (AY922177), and Uncultured methylibium sp. (KF469187) these four kinds of bacteria exist alone in Acinetobacter sp. treatment group, while Uncultured pseudomonas sp. (JQ279040) existed separately in the Bacillus subtilis treatment group. At the same time, it was found that the abundance of Bacillus firmus strain sctcc471(HQ622343), Uncultured bacterium clone (FJ719098), and Uncultured alpha proteobacterium (KM978288) in rhizosphere soil of Bacillus subtilis treatment group was much higher than that of Acinetobacter sp.treatment group and clear water control group on the 40th day.To sum up, root irrigation with biological agents of Bacillus subtilis and Acinetobacter sp. can affect the structure and abundance of microorganisms in crop rhizosphere and promote the colonization of microflora and the growth of pepper seedlings to a certain extent. The high abundance of five bacterial strains, Acinetobacter sp. (AM295822), Uncultured pseudomonas sp. (JQ279040),Bacillus firmus strain sctcc471(HQ622343),Uncultured bacterium clone(FJ719098),and Uncultured alpha proteobacterium (KM978288), may play a positive role in promoting the growth of pepper seedlings, which lays a foundation for further research and development of biological agents, and provide new ideas and methods for environmental control and ecological security protection.

HomePlant DiseaseVol. 104, No. 9First Report of Leaf Blight on Chinese Fir (Cunninghamia lanceolata) Caused by Bipolaris setariae in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Leaf Blight on Chinese Fir (Cunninghamia lanceolata) Caused by Bipolaris setariae in ChinaWen-Li Cui, Jin-Yue Bian, De-Wei Li, Jun-Wei Wang, and Lin HuangWen-Li CuiCo-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, Jiangsu 210037, ChinaSearch for more papers by this author, Jin-Yue BianCo-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, Jiangsu 210037, ChinaSearch for more papers by this author, De-Wei Lihttp://orcid.org/0000-0002-2788-7938The Connecticut Agricultural Experiment Station Valley Laboratory, Windsor, CT 06095, U.S.A.Search for more papers by this author, Jun-Wei WangKunyushan Forest Farm, Yantai, Shandong 264100, ChinaSearch for more papers by this author, and Lin Huang†Corresponding author: L. Huang; E-mail Address: lhuang@njfu.edu.cnhttp://orcid.org/0000-0001-7536-0914Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, Jiangsu 210037, ChinaSearch for more papers by this authorAffiliationsAuthors and Affiliations Wen-Li Cui1 Jin-Yue Bian1 De-Wei Li2 Jun-Wei Wang3 Lin Huang1 † 1Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, Jiangsu 210037, China 2The Connecticut Agricultural Experiment Station Valley Laboratory, Windsor, CT 06095, U.S.A. 3Kunyushan Forest Farm, Yantai, Shandong 264100, China Published Online:1 Jul 2020https://doi.org/10.1094/PDIS-12-19-2685-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Chinese fir (Cunninghamia lanceolata [Lamb.] Hook) is widely cultivated in China, contributing ∼40% to timber production in southern China. In December 2017, a leaf blight disease was found on Chinese fir in the National Forest Park of Kunyushan (E 121°46′, N 37°15′) in Yantai, Shandong. It mainly infected Chinese fir, exhibiting brown to brownish-red with one to three dark brown bands across the leaves. It started at the tips and later covered the whole leaf. Incidence was 9.4%. Symptomatic leaves were collected and surface sterilized. Forty cuttings (∼0.5 × 0.5 cm) from lesion margins were placed on 2% PDA in Petri plates and incubated at 25 ± 2°C for 7 days. Twenty-eight isolates were single spored and stored in the Forest Pathology Laboratory at Nanjing Forestry University. They were grown on PDA for 7 days and exposed to fluorescent light for another 5 days to stimulate sporulation. Conidia of each isolate were individually collected in sterile ddH2O and used for plant inoculations (1 × 105 spores/ml). Healthy 1-year-old detached leaves collected in the field and 10-month-old seedlings generated from tissue culture were surface sterilized using 75% ethanol and inoculated with 5 µl of the conidial suspension. Sterile ddH2O was used on control leaves. Inoculated leaves and plants were kept in a moist chamber at 25 ± 2°C under a 12-h photoperiod. The experiment was repeated twice, with at least six replicates for each treatment. At 7 days postinoculation, detached leaves inoculated with isolate XXG7 showed brown necrotic lesions of 1.4 ± 0.6 cm average length. Similar necrotic lesions were observed on seedlings inoculated by XXG7. No lesions were found on plants inoculated by other fungal isolates and ddH2O. XXG7 was reisolated from the margins of the lesions and used to fulfill Koch’s postulates and for species identification. Colonies of XXG7 grown on PDA were round, with an average mycelial growth rate of 1.6 ± 0.4 cm/day. Aerial mycelium was dense, felted, and olive. The fungus produced black pigments. The vegetative hyphae were septate, branched, light brown to brown, 1.9 to 3.4 μm in width. When the colonies were exposed to fluorescent light for 3 days, conidia and conidiophores were produced. Conidiophores were solitary, brown, rarely branched, multiseptate, flexuous, and geniculate at the upper parts. Conidiophores were 66.8 to 355.6 × 3.5 to 6.7 μm, and the terminal conidiogenous cells were 6.6 to 22.3 μm long (n = 30). Conidia were usually long, oval, straight, smooth, brown to dark brown. Most conidia were 4- to 9-distoseptate and 27.1 to 75.4 × 12.6 to 21.3 μm (mean = 53 × 16.3 μm [n = 60]). These morphological characteristics were similar to Bipolaris setariae (Manamgoda et al. 2014; Shoemaker 1959). The taxonomy was further studied using molecular identification. The ITS region of rDNA, glyceraldehyde-3-phosphate dehydrogenase (GPD) gene, and translation elongation factor-alpha (tef-1α) gene of XXG7 were amplified with primer sets ITS1/ITS4, GDP1/DPG2, and EF1-983/EF1-2218R, respectively (Raza et al. 2019). ITS (MT032396), GPD (MT036970), and tef-1α (MT036971) sequences were deposited in GenBank. The ITS sequence showed 99.8% identity to B. setariae strain LC12047 (MN215632.1); GPD sequence 100% similarity to strain LC12047 (MN264068.1); and tef-1α sequence 99.9% similarity to strain LC12047 (MN263926.1). Phylogenetic analysis using concatenated sequences of ITS, GPD, and tef-1α also showed that XXG7 clustered monophyletically with strains of B. setariae and was supported with a high bootstrap value (73%). Based on morphological and phylogenetic data, XXG7 was identified as B. setariae. B. setariae is an important pathogen on lawn grasses, gramineous crops, and other plants (Manamgoda et al. 2014; Raza et al. 2019). This is the first report of B. setariae causing leaf blight on Chinese fir in China. This discovery will facilitate further studies as well as monitoring and control of the disease in the future.The author(s) declare no conflict of interest.References:Manamgoda, D. S., et al. 2014. Stud. Mycol. 79:221. https://doi.org/10.1016/j.simyco.2014.10.002 Crossref, ISI, Google ScholarRaza, M., et al. 2019. Fungal Divers. 99:1. https://doi.org/10.1007/s13225-019-00434-5 Crossref, ISI, Google ScholarShoemaker, R. A. 1959. Can. J. Bot. 37:879. https://doi.org/10.1139/b59-073 Crossref, Google ScholarThe author(s) declare no conflict of interest.Funding: Funding was provided by National Key R & D Program of China (2017YFD0600102) and National Natural Science Foundation of China (31870631).DetailsFiguresLiterature CitedRelated Vol. 104, No. 9 September 2020SubscribeISSN:0191-2917e-ISSN:1943-7692 DownloadCaptionRipening strawberry infected by Colletotrichum acutatum (M. Dowling et al.). Photo credit: M. Dowling. Diaporthe leaf spot caused by Diaporthe humulicola on hop cone tissue (E. Allan-Perkins et al.). Photo credit: M. Salvas. Metrics Article History Issue Date: 26 Aug 2020Published: 1 Jul 2020First Look: 5 Apr 2020Accepted: 2 Apr 2020 Pages: 2523-2523 Information© 2020 The American Phytopathological SocietyFundingNational Key R & D Program of ChinaGrant/Award Number: 2017YFD0600102National Natural Science Foundation of ChinaGrant/Award Number: 31870631Keywordsasexual fungiPleosporalesforestetiologyThe author(s) declare no conflict of interest.Cited byA real‐time PCR for detection of pathogens of anthracnose on Chinese fir using TaqMan probe targeting ApMat gene14 November 2022 | Pest Management Science, Vol. 79, No. 3Cochliobolus setariae (millet blight)CABI Compendium, Vol. CABI Compendium