Sinensis In China Research Articles

Screening and Site Adaptability Evaluation of Qi-Nan Clones (Aquilaria sinensis) in Southern China

In recent years, plantations of Aquilaria sinensis in China have been dominated by Qi-nan, yet there remains limited research on the growth evaluation and breeding of these clones. In this study, a multi-point joint variance analysis, an additive main effect and multiplicative interaction (AMMI) model, a weighted average of absolute scores (WAASB) stability index, and a genotype main effect plus a genotype-by-environment interaction (GGE) biplot were used to comprehensively analyze the yield, stability, and suitable environment of 25 3-year-old Qi-Nan clones from five sites in southern China. The results showed that all the growth traits exhibited significant differences in the clones, test sites, and interactions between the clones and test sites. The phenotypic variation coefficient (PCV) and genetic variation coefficient (GCV) of the clones’ growth traits at the different sites ranged from 16.56% to 32.09% and 5.24% to 27.06%, respectively, showing moderate variation. The medium–high repeatability (R) of tree height and ground diameter ranged from 0.50 to 0.96 and 0.69 to 0.98, respectively. Among the clones, Clones G04, G05, G10, G11 and G13 showed good growth performance and could be good candidates for breeding. Environmental effects were found to be the primary source of variation, with temperature and light primarily affecting growth, while rainfall influenced survival and preservation rates. Yangjiang (YJ) was found to be the most suitable experimental site for screening high-yield and stable clones across the different sites, whereas the tree height and ground diameter at the Chengmai (CM) site were significantly higher than at the other sites, and the Pingxiang (PX) and Zhangzhou (ZZ) sites showed poor growth performance. The findings suggest that Qi-nan clones are suitable for planting in southern China. There were also abundant genetic variations in germplasm resources for the Qi-nan clones. The five selected clones could be suitable for extensive planting. Therefore, large-scale testing is necessary for determining genetic improvements in Qi-nan clones, which will be conducive to the precise localization of their promotion areas.

First Report of Didymella pomorum Causing Leaf Blight on Angelica sinensis in China.

Angelica sinensis (Oliv.) Diels, is a perennial herbaceous plant of the Umbelliferae family. It has a long history of cultivation and is highly valued as a traditional Chinese medicine in China (Zhang et al. 2012). In September 2023, leaf blight on A. sinensis with an average disease incidence of 56% was recorded in an approximately 6.7-ha production field in Lijiang, Yunnan province, China (26.8215°N, 100.2369°E). At first, small, chlorotic lesions appeared on the leaves. They subsequently increased in density and gradually merged, causing leaves to yellow and wither. Ultimately the blight casused death of the entire foliage. In order to identify the causal agent, cross-sectional segments (5×5 mm2) were cut from the edge of leaf lesions, surface disinfected with a 1% sodium hypochlorite solution for 3 min and rinsed three times with sterile distilled water. They were subsequently placed on potato dextrose agar (PDA) plates and incubated for 3 days under a 12-h photoperiod at 28℃. A total of ten isolates with similar morphological characteristics were obtained by single spore isolation. After 10 days of incubation on PDA, the colony morphology of these isolates was characterized by a brownish central area with a white edge. Aged colonies became wrinkled in the center of the colony. Conidia (n = 30) were elliptical and brown, with a size range of 4.11 to 6.55 μm (average 5.37±0.74 μm) × 3.17 to 4.62 μm (average 3.92±0.43 μm). Chlamydospores (n = 30) formed chains in series, spherical or elliptical in shape, ranging from yellow-brown to dark brown, with a size range of 12.30 to 13.70 μm (average 12.98±0.46 μm) × 4.20 to 5.30 μm (average 4.63±0.26 μm). The nuclear ribosomal internal transcribed spacer region (ITS), the second largest subunit of RNA polymerase II (RPB2), and the 28S nuclear ribosomal large subunit rRNA (LSU) region of two isolates were amplified with the primer pairs ITS1/ITS4 (White et al. 1990), fRPB2-5F/fRPB2-7cR (Liu et al. 1999), and LR0R/LR5 (Schoch et al. 2012), respectively. These amplicons were sequenced bidirectionally and assembled. The two isolates produced the same nucleotide sequences, and the sequences of a representative isolate (AsDp1) were deposited in GenBank. BLASTn analyses showed that the ITS (PP510616), RPB2 (PP526010), and LSU (PP550143) sequences of isolate AsDp1 were 100%, 99.66%, and 100% identical with those of Didymella pomorum ex-type isolate CBS 354.52 (MH857081, KT389616, and MH868616), respectively. A phylogenetic tree was constructed based on the ITS, RPB2, and LSU concatenated nucleotide sequences using the maximum likelihood method in MEGAX. Isolate AsDp1 was clustered with four D. pomorum isolates. According to the morphological and nucleotide sequences analyses, isolate AsDp1 was identified as D. pomorum (Chen et al. 2015). To determine pathogenicity, 1-year-old A. sinensis plants (approximately 20 cm tall) grown in 7-liter pots filled with sterilized field soil were sprayed until runoff with a 1×106 conidia/ml suspension of isolate AsDp1 onto the foliage, while control plants were sprayed with sterile water. All plants were cultivated under a 12-h photoperiod at 25℃. The pathogenicity tests were performed in triplicate with ten plants in each treatment. After fifteen days, numerous chlorotic lesions appeared on the leaves of all inoculated plants. The symptoms were similar to those found on naturally infected plants in the field, while the control plants remained asymptomatic. Subsequently, D. pomorum was reisolated from the diseased leaves, and the identity was confirmed based on its ITS sequence and morphological characteristics. D. pomorum causing stem canker on Rosa spp. was reported in Canada (Ilyukhin 2022). To our knowledge, this is the first report of D. pomorum causing leaf blight on A. sinensis in China. This etiological finding will potentially pave the way for the development of control strategies of this disease.

Comparative transcriptome analysis reveals the differences in wound-induced agarwood formation between Chi-Nan and ordinary germplasm of Aquilaria sinensis

Agarwood is a rare and valuable heartwood derived from Aquilaria sinensis in China. Compared with ordinary germplasm, Chi-Nan, a special germplasm of A. sinensis, has a better agarwood-producing capacity. However, the mechanisms underlying their different qualities remain poorly characterized. Here, a comparative transcriptome analysis of Chi-Nan and ordinary A. sinensis was carried out to investigate the wound responses of both germplasms. A total of 198.19 Gb of clean data were obtained with an average of 6.61 Gb of clean reads for each sample. By comparing with their control groups, more differentially expressed genes (DEGs) were observed in Chi-Nan germplasm. Kyoto Encyclopedia of Genes and Genomes (KEGG) and expression profile analysis suggested that Chi-Nan possesses a stronger ability to respond to wounding. Furthermore, the enrichment of biosynthetic pathways related to sesquiterpenes and 2-(2-phenylethyl) chromones (PECs) were more significant in Chi-Nan than in ordinary germplasm, and related genes showed significantly higher up-regulation in Chi-Nan after wounding. Sixteen candidate genes presumably involved in biosynthesis of agarwood components were identified and found to exhibit higher up-regulation in Chi-Nan than in ordinary germplasm in response to wounding. Overall, these results are helpful in explaining reasons for the higher agarwood-producing properties of Chi-Nan, and contribute to a further understanding of the mechanism of agarwood formation in A. sinensis.

Evaluation of Cordyceps sinensis Quality in 15 Production Areas Using Metabolomics and the Membership Function Method.

Cordyceps sinensis is a precious medicinal and edible fungus, which is widely used in body health care and disease prevention. The current research focuses on the comparison of metabolite characteristics between a small number of samples and lacks a comprehensive evaluation of the quality of C. sinensis in a large-scale space. In this study, LC-MS/MS, principal component analysis (PCA), hierarchical cluster analysis (HCA), and the membership function method were used to comprehensively evaluate the characteristics and quality of metabolites in 15 main producing areas of C. sinensis in China. The results showed that a total of 130 categories, 14 supercategories, and 1718 metabolites were identified. Carboxylic acids and derivatives, fatty acyls, organo-oxygen compounds, benzene and substituted derivatives, prenol lipids, and glycerophospholipids were the main components of C. sinensis. The HCA analysis and KEGG pathway enrichment analysis of 559 differentially accumulated metabolites (DAMs) showed that the accumulation models of fatty acids and conjugates and carbohydrates and carbohydrate conjugates in glycerophospholipid metabolism and arginine and proline metabolism may be one of the reasons for the quality differences in C. sinensis in different producing areas. In addition, a total of 18 biomarkers were identified and validated, which had a significant discrimination effect on the samples (p < 0.05). Overall, YS, BR, and ZD, with the highest membership function values, are rich and balanced in nutrients. They are excellent raw materials for the development of functional foods and provide scientific guidance for consumers to nourish health care.

Characterizing the Pathogenicity and Mycotoxin Production Capacity of Fusarium spp. Causing Root Rot of Angelica sinensis in China.

Root rot is a very destructive soil-borne disease, which severely affects the quality and yield of Angelica sinensis in major planting areas of Gansu Province, China. Twelve Fusarium strains were identified from root rot tissue and infected soil in the field by comparing each isolate strain internal transcribed spacer, translation elongation factor 1-α sequence and RNA polymerase second largest subunit gene with the sequences of known fungal species in the NCBI database. Of these isolates, four were F. acuminatum, followed by three F. solani, two F. oxysporum, and one each of F. equiseti, F. redolens, and F. avenaceum. Under greenhouse conditions, pathogenicity testing experiment was carried out using five strains: two F. acuminatum, one F. solani, one F. oxysporum, and one F. equiseti. Among them, the incidence of F. acuminatum-induced root rot on A. sinensis was 100%; hence, it was the most aggressive. Liquid chromatography was used to show that F. acuminatum could produce neosolaniol (NEO), deoxynivalenol, and T-2 toxins. Of these, the level of NEO produced by F. acuminatum was high compared with the other two toxins. By isolating Fusarium spp. and characterizing their toxin-producing capacity, this work provides new information for effectively preventing and controlling A. sinensis root rot in the field as well as improving the quality of its medicinal materials.

First Report of Botrytis cinerea Causing Gray Mold on Tea (Camellia sinensis) in China.

Tea (Camellia sinensis (L.) O. Kuntze) is an important economic crop cultivated in China. In March 2019, leaf blight symptoms were observed on tea trees (C. sinensis cv. Fuding-dabaicha) in Enshi City (30º02'66" S, 109º01'56" W), Hubei Province (Fig. 1A). The disease occurred on the senescent flowers and mature leaves. The average disease incidence was approximately 10% of plants in the surveyed 30 ha tea garden. Initial symptoms consisted of brown lesions, which expanded and became covered with gray mold, followed by abscission. To identify the pathogen, 20 symptomatic leaves were collected and 0.5 cm2 diseased leaf pieces were excised and surface sterilized by immersion in 1.5% sodium hypochlorite (NaOCl) for 2 min, and rinsed three times in sterilized distilled water. The leaf pieces were allowed to dry, placed on potato dextrose agar (PDA) plates, and incubated at 20°C under 12h fluorescent light. The fungus formed gray to grayish brown colonies (Fig. 1B) and produced conidia after 1 week. The isolate was purified by single spore isolation. The conidia were one-celled, ellipsoid or ovoid, almost colorless, with a size range of 5.3 to 10.24 × 5.2 to 8.1 μm (n = 50) (Fig. 1C). The sclerotia produced in culture were black, round or irregular in shape and 1.3 to 2.2 × 2.0 to 3.3 mm (average 1.8 × 2.3 mm) in size. Morphological characteristics of these isolates matched the description of Botrytis spp. (Hong et al. 2001). Representative isolate LCHM was selected for molecular identification based on DNA sequencing of the ITS region of rDNA and three nuclear protein-coding genes (G3PDH, HSP60 and RPB2) (Staats et al. 2005). BLAST analysis showed that isolate LCHM (GenBank Acc. Nos. MN448502, MN448500, MN433708 and MN448501 for ITS, G3PDH, HSP60 and RPB2, respectively) shared 99 to 100% identity with B. cinerea (GenBank Acc. Nos. MH316147.1, MG846500.1, MG846504.1 and MG846510.1, respectively), which suggested that isolate LCHM belongs to B. cinerea. This identification was further confirmed by phylogenetic analysis based on combined DNA sequence data of G3PDH, HSP60 and RPB2 (Fig. 2). For the pathogenicity test, needle-wounded, attached leaves on 1-year-old C. sinensis cv. Fuding-dabaicha plants were inoculated with mycelial plugs (6 mm diameter from 2-day-old PDA cultures) or a conidial suspension (20 μL, 1×106 conidia/ml in half strength potato dextrose broth) of isolate LCHM on three plants per treatment (six leaves). The control treatments were also wounded, but only treated with agar plugs or half strength PDB. All inoculated plants and controls were incubated in a growth chamber (20°C, 90 ± 10% RH). Leaves inoculated with mycelial plugs, whether wounded or not, showed brown necrotic lesions around the agar plugs after 2 days (Fig. 1D, E), whereas conidial inoculations of wound sites showed necrotic lesions after 6 days (Fig. 1 F, G). Non-wounded leaves inoculated with conidia and all the control treatments remained symptomless. B. cinerea was reisolated from the inoculated leaves and isolates were morphologically similar to the original cultures. Gray mold of C. sinensis caused by B. cinerea has been recorded in Japan, Turkey, and Brazil (Hamaya 1981, Aziz and Harun 2010, Pereira and Mio, 2020). To our knowledge, this is the first report of gray mold caused by B. cinerea on C. sinensis in China. B. cinerea may cause economic losses of tea, therefore, more surveys in other tea-growing regions should be done to specifically search for this disease.

First Report of Postharvest Fruit Rot on Gannan Navel Orange Caused by Diaporthe unshiuensis in Jiangxi Province, China.

The Gannan navel orange (Citrus sinensis Osbeck cv. Newhall) is one of the most widely planted citrus fruit cultivars in Ganzhou City, Jiangxi Province, China. A Gannan navel orange was harvested from an orchard in Yudu County, Ganzhou City, Jiangxi Province, China, in October 2022 (25.95N, 115.41E). Approximately 5% of the fruit rotted after being stored at room temperature for about two weeks.Infected fruits appear brown and rotted with slightly indented edges. Initially symptoms of infected fruits was small circular, light brown, which the rot expands, slightly water-stained halo circle with slightly indented edges. The surface of 10 infected fruits was sterilized with 75% ethanol, and the lesion edge was cut into 5-mm-diameter pieces, and the pieces were then placed on potato dextrose agar (PDA) and incubated at 25°C for five days. A total of eight morphologically similar isolates were obtained. PDA results showed dense white and fluffy aerial mycelia in the center of colonies with sparser edges. Two types of conidia were produced; the alpha conidia were hyaline, ellipsoidal or clavate, and aseptate, with 2 oil drops, 4.8 to 7.5 × 2.1 to 2.7 μm (n=30). The beta conidia were hyaline, aseptate, filiform, smooth, straight to sinuous, 16.9 to 27.5 × 1.3 to 1.6 μm (n=30). These isolates exhibit morphological characteristics similar to those of the Diaporthe genus. Genomic DNA of two representative isolates (JFRL-03-1130 and JFRL-03-1131) was extracted for further confirmation. The internal transcribed spacer (ITS) region, beta-tubulin (TUB), calmodulin (CAL), partial translation elongation factor 1-alpha (TEF1-α), and histone H3 (HIS3) genes were amplified and sequenced using primers ITS1/ITS4, Bt2a/Bt2b, CAL228F/CAL737R, EF1-728F/EF1-986R, and CYLH3F/H3-1b, respectively (Udayanga et al. 2015). These nucleotide sequences were deposited into the GenBank database with accession numbers OQ691637-OQ691638 (ITS), OQ701022-OQ701023 (TUB), OQ701016-OQ701017 (CAL), OQ701018-OQ701019 (TEF1-α) and OQ701020-OQ701021 (HIS3). The maximum likelihood analyses were performed for the combined ITS, TEF1-a, TUB, HIS3, and CAL data set using Phylosuite V1.2.2 (Zhang et al. 2020). The phylogenetic tree showed that the two isolates clustered with D. unshiuensis in a clade with 100% bootstrap support. Therefore, the fungus was identified as D. unshiuensis based on morphological and molecular characteristics. To evaluate pathogenicity, a sterile scalpel was used to wound 10 surface-sterilized fruits, and a 5-mm-diameter mycelial plug of the isolate JFRL 03-1130, cultured on PDA at 25℃ for 7-days, was put on the wound. Another set of 10 fruits was similarly inoculated with sterile agar plugs as controls. The fruits were cultured at 25°C and 85% relative humidity, and the test was repeated twice. These fruits inoculated with D. unshiuensis showed similar rot symptoms after 10 days, while the control group remained symptomless. In order to prove Koch's postulates, the pathogen was re-isolated from the inoculated fruits and confirmed as D. unshiuensis by molecular techniques, but never from the control fruits. Diaporthe unshiuensis has been reported as an endophyte associated with citrus and a pathogen that causes melanose disease in citrus (Chaisiri et al. 2020; Huang et al. 2015). However, to the best of our knowledge, this is the first reported case of D. unshiuensis causing postharvest fruit rot on Citrus sinensis. In the past, D. sojae has also been reported causing postharvest fruit brown rot disease on Citrus sinensis in China (Xiao, et al. 2023); Therefore, managers should pay more attention to postharvest fruit rot disease caused by Diaporthe species and implement storage strategies to control and reduce losses.

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Wisteria sinensis in China.

Wisteria (Wisteria sinensis) is a well-known ornamental plant for environmental protection in the garden, which also has a high value for medicinal use. In December 2021, leaf spots were observed on W. sinensis plants growing on the campus of Jiangxi Agricultural University in Nanchang, Jiangxi Province (28.45° N, 115.49° E), with a incidence rate of 40% plants were infested (n = 100 investigated plants). Initially leaf spots were small and pale brown (Approx. 2 mm in diameter), which gradually expanded into round or irregular dark brown spots as disease progressed, and lesions developed greyish-white necrotic tissues in the center at later stages, eventually causing the leaves to rot. To isolate the pathogen, tissues (5 × 5 mm) at the margin of lesions were cut from ten symptomatic leaves, surface disinfected with 75% ethanol for 30 s followed by 2% sodium chloride (NaClO) for 1 min, rinsed three times with sterile distilled water, and the dried tissues were cultured on potato dextrose agar (PDA) at 28 ± 1℃ in darkness for 3 days. After culture purification, five isolates were obtained and the representative single spore isolate (ZTTJ1) was used for subsequent identification tests. After 10 days of incubation on PDA medium, colonies had dense aerial mycelium with a gray center and dark gray-green mycelium outward, with orange-red conidial masses distributed in a ring on the surface. The underside of the colonies was light gray to dark gray. Conidia were cylindrical, with ends obtuse-rounded, 11.83 to 20.74 × 3.34 to 5.33 μm (av=16.11 μm × 4.26 μm, n = 50) in size. These morphological characteristics were consistent with Colletotrichum gloeosporioides (Shi et al, 2019). Six conserved regions of isolate (ZTTJ1), internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), β-tublin (TUB), actin (ACT), and chitin synthase 1 (CHS1) gene regions were amplified using ITS1/ITS4 (Gardes et al, 1993), GDF/GDR (Templeton et al, 1992), CL1C/CL2C (Li et al, 2018), Bt2a/Bt2b (Prihastuti et al, 2009), ACT-512F/ACT-783R and CHS-79F/CHS-345R (Carbone et al, 1999) primers, respectively. Using BLAST, ITS, GAPDH, CAL, TUB, ACT, and CHS1 gene sequences (GenBank Accession No. OP703312, OP713773, OP713775, OP713776, OP713772, OP713774, respectively) were over 99% identical to C. gloeosporioides (GenBank Accession No. MK967281, MH594288, MT449307, MN624110, MN107239 and MN908602, respectively). A maximum likelihood (ML) phylogenetic analysis based on ITS-ACT-GAPDH-CHS1-CAL-TUB2 sequences using MEGA7.0, placed isolate (ZTTJ1) within C. gloeosporioides. To complete Koch's postulates, 10 μL spore suspension (1.0 × 106 conidia/mL) of ZTTJ1 (7-day-old culture on PDA medium) was dropped onto 10 leaves wounded with a sterilized needle and 10 non-wounded leaves, respectively. Ten wounded leaves were inoculated with sterile water as controls. All leaves were incubated at 28 ± 1 °C and 90 % relative humidity (12 h/12 h light/dark). After 7 days, all wounded leaves inoculated with C. gloeosporioides developed symptoms as previously observed, while the control and non-wounded leaves remained healthy. The fungus re-isolated from the inoculated leaves were identified as C. gloeosporioides by morphological and molecular identification; the pathogen causing disease in W. sinensis was determined to be C. gloeosporioides. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose on W. sinensis in China. This work has identified the pathogenic species of the disease, which helps to take targeted measures to control its spread, providing a basis for the prevention and treatment of the disease.

First report of Fusarium solani causing wilt disease on Gleditsia sinensis in Tianjin, China.

The Gleditsia sinensis Lam. widely grown in China is a perennial plant with medicinal properties (Zhang et al. 2016). Since 2019, the leaves of G. sinensis have exhibited yellowing and wilting, and the plants have gradually become stunted and dead in Taifeng park of Binhai New Area in Tianjin (39.02° N; 117.65° E). In this park, there are two types of G. sinensis, one is with round branch thorns, the other is with flat branch thorns. The G. sinensis with round branch thorns did not grow well and almost all plants had disease symptoms. The samples were collected on October, 2021 and deposited in Plant Disease Laboratory of Tianjin Agricultural University under accession no. PATAU211018. The disease symptoms consisted of foliage wilt (Figure 1A), plant drying and vascular tissue discoloration (Figure 1B). The stem sections from different plants were surface-disinfested in 0.6% NaClO, wiped with 75% ethanol and rinsed with sterile water. Thirty tissue samples were placed on Potato Dextrose Agar (PDA) medium and cultured at 28℃ for 7 days (Uppala et al. 2013). Thirty fungal isolates with the same morphological characteristics were obtained from the samples. Five representative isolates (PATAU211018-05, PATAU211018-07, PATAU211018-10, PATAU211018-12 and PATAU211018-21) were collected and purified using the single-spore method (Li et al. 2022). Colonies of the five isolates on PDA grew in a circular shape and showed abundant white densely fluffy aerial mycelium (Figure 1C). Morphological characteristics included septate and hyaline hyphae, long cylindrical monophialides (Figure 1D), macroconidia (Figure 1E) and microconidia (Figure 1F). Macroconidia were falcate, 2-5-septate, hyaline, 18-40 × 4-6 μm (n = 50). Microconidia were hyaline, oblong, 0-1-septate, 5-14 × 2-6 μm (n = 50). These morphological characteristics were consistent with the description of Fusarium solani. (Chitrampalam et al. 2018). PATAU211018-12 was randomly chosen for molecular analysis as the representative isolate given the similarity of these isolates. For further identification, the genomic DNA of isolate PATAU211018-12 was extracted. The fragments of internal transcribed spacer (ITS), translation elongation factor 1α (EF1α) gene and RNA polymerase II subunit (RPB2) were amplified and sequenced (O'Donnell et al. 2008; Carbone et al. 1999). The sequences of ITS, EF1α, and RPB2 of PATAU211018-12 were deposited in GenBank under the accession no. of OP735578, ON630412 and OP746032, respectively. Phylogenetic trees were constructed in MAGA X (Kumar et al. 2018) using the neighbor-joining (NJ) method based on the concatenated sequences of ITS, EF1α, and RPB2 (Figure 2). The isolate (PATAU211018-12) grouped with F. solani (JS-169) with a bootstrap value of 100 in the phylogenetic tree. The morphology and multi-gene phylogenetic analysis indicated that the new isolate is F. solani. Pathogenicity tests were carried out on one-year-old G. sinensis seedlings with round branch thorns (n=6). The F. solani isolate PATAU211018-12 was cultured in Potato Dextrose Broth (PDB) at 28°C on a shaker at 150 rpm for 5 days. Mycelia were filtered through four layers of sterilized lens paper and the conidia were obtained for pathogenicity tests. G. sinensis was infected by F. solani through root soaking method. The roots were inoculated by dipping in conidial suspension with the concentration of 107 conidia/mL for 30 minutes. Control plants (n=6) were treated with distilled water. Plants were in pots indoors at 25℃. At 20 days after inoculation, the leaves of inoculated plants were chlorotic and wilted (Figure 1G), symptoms similar to those observed in the park. In contrast, the leaves of control plants were symptomless (Figure 1H). The pathogenicity assay was repeated three times. The fungal isolate was re-isolated from the disease tissues and verified as F. solani based on morphology and molecular character (ITS, EF1α and RPB2). F. solani has been reported as pathogens on many plants, such as Eriobotrya japonica (Wu et al. 2021), Fragaria × ananassa (Pastrana et al. 2014), Gastrodia elata (Li et al. 2022) and Hedysarum boreale (Uppala et al. 2013). To our knowledge, this is the first report of F. solani causing disease on G. sinensis in China. Identification of F. solani as a disease agent in G. sinensis will assist in disease management for this important tree crop.

Current Disease Threats for Cultivated Crab Eriocheir sinensis in China

The Chinese mitten crab, Eriocheir sinensis, is a commercially important crustacean in China due to its great commercial value and compatibility in a variety of aquaculture systems. However, increases in its production have been accompanied by the emergence of various diseases affecting yield, profit, and trading potential. In this study, we review the pathogenic agents associated with E. sinensis since the start of its commercial culture. The history of crab cultivation implies that increased pathogen transfer can occur as E. sinensis aquaculture grows because polyculture of E. sinensis with other aquaculture species is a prevalent practice. With this in mind, a special focus of this review is placed on pathogens that were initially discovered in other crustacean species but have since been demonstrated to infect and cause disease in E. sinensis. We expect that this review will not only offer recommendations for disease management in the E. sinensis aquaculture sector but will also advance other crustacean cultivation.

The malaria transmission in Anhui province China.

Plasmodium vivax and Plasmodium falciparum cases have opposite trends in Anhui China in the past decade. Long term and seasonal trends in the transmission rate of P. falciparum in Africa has been well studied, however that of P. vivax transmitted by Anopheles sinensis in China has not been investigated. There is a lot of work on the relationship between P. vivax cases and climatic factors in China, with sometimes contradicting results. However, how climatic factors affect transmission rate of P. vivax in China is unknown. We used Anhui province as an example to analyze the recent transmission dynamics where two types of malaria have been reported with differing etiologies. We examined breakpoints of the P. vivax and P. falciparum malaria long term dynamics in the recent decade. For locally transmitted P. vivax malaria, we analyzed the transmission rate and its seasonality using the combined human and mosquitos SIR-SI model with time-varied mosquito biting rate. We identified the effects of meteorological factors on the seasonality in transmission rate using a GAM model. For the imported P. falciparum malaria, we analyzed the potential reason for the observed increase in cases. The breakpoints of P. vivax and P. falciparum dynamics happened in a same year, 2010. The seasonality in the transmission rate of P. vivax malaria was high (42.4%) and was linearly associated with temperature and nonlinearly with rainfall. The abrupt increase in imported P. falciparum cases after the breakpoint was significantly related to the increased annual Chinese investment in Africa. Under the conditions of the existing vectors of malaria, long-term trends in climatic factors, and increasing trend in migration to/from endemic areas and imported malaria cases, we should be cautious of the possibility of the reestablishment of malaria in regions where it has been eliminated or the establishment of other vector-borne diseases.

Effects of morphological traits on living body weight of wild Cyclina sinensis in different geographical populations

Eleven coastal geographical populations of wild Cyclina sinensis in China were collected in February 2020, and the effects of four morphological traits (shell length; shell height; shell width; external ligament length) on one weight trait (living body weight) were studied by correlation analysis, path analysis, determination coefficient analysis, and regression analyses. The statistical results showed that the coefficient of body weight variation was generally greater than morphological traits(P＜0.05). The correlation analysis results showed that the coefficient of correlation between morphological traits (except for external ligament length) and body weight are significantly positive (P＜0.05) in all populations. Based on the results of path analysis and determination coefficient analysis, shell length has the greatest direct effect on body weight in the Yancheng population; shell height has the greatest direct effect on body weight in Dandong, Fuzhou and Tangshan populations; shell width has the greatest direct effect on body weight in Zhanjiang, Wenzhou, Dongtai, Ningbo, Tianjin, Dongying, and Wanning populations. Multiple regression equations were obtained with body weight as the dependent variable, shell length, height, and width and external ligament length as independent variables. The results of systematic clustering showed that there are no apparent geographical differentiation characteristics among eleven geographical populations in morphology. This study provided a scientific basis for selective and genetic breeding and can guide the development and utilization of wild C. sinensis seed resources.

Fine Structure of the Compound Eyes of Male and FemaleHeortia vitessoidesMoore (Lepidoptera: Crambidae)

AbstractHeortia vitessoides is a destructive pest of the economically important plant Aquilaria sinensis in China. We investigated the external morphology and ultrastructure of H. vitessoides compound eyes using scanning and transmission electron microscopy. We found that H. vitessoides has superposition eyes, made up of a mean of 3,671 and 3,428 ommatidia in males and females, respectively. The mean ommatidial diameter for males and females was 18.87 and 16.81 µm, respectively. Each ommatidium was capped by a convexly curved corneal facet lens of about 13.85 µm thickness. Corneal nipples measuring 226.36 nm in height in males and 295.48 nm in females, covered the surface of the eyes. Heortia vitessoides eyes are eucone, with each ommatidium having a crystalline cone comprising four cone cells, surrounded by two primary pigment cells. The centrally fused rhabdom of each ommatidium was formed by 8–11 of retinal cells, somewhat more than are typically found in Lepidoptera. The relatively large size of the compound eyes, the large number of ommatidia, large number of retinula cells, and high corneal nipples all suggest that H. vitessoides eyes are adapted to optimize visual sensitivity under dim light conditions.

Root Rot of Angelica sinensis Caused by Clonostachys rosea and Fusarium acuminatum in China.

Angelica sinensis is a perennial plant in the Apiaceae and a source of one of the most important traditional medicines in China. This medicinal herb has been mainly cultivated in Gansu, Sichuan, Yunnan, Shanxi, Hubei, and Qinghai Provinces. In June 2020, angelica plants in a field in the village of Sada, Minhe County, Haidong city, in Qinghai Province (102°83'00.00″E, 36°31'93.00″N, elevation:2437 m), displayed symptoms of yellowing, stunting, root rot, and wilting（Fig. S1）. Severe brown discoloration of vascular tissue in the stems of infected plants was observed. Root rot seriously affected the quality and yield of A. sinensis. The incidence of root rot in the fields reached 15% in 2020 and 18% in 2021. Fungi were isolated from diseased roots using a single spore method. The identity of isolates was confirmed by sequencing the nuclear ribosomal internal transcribed spacer (ITS) using the primers ITS1/ITS4 (White et al, 1990) and the translation elongation factor 1-alpha (TEF1-α) gene using the primers EF-1/EF-2 (O'Donnell et al. 1998). BLAST analysis of the ITS and TEF1-α sequences of isolates DG-1, DG-1-1, DG-1-2, DG-2, DG-2-1, DG-5, and DG-5-1 in the NCBI database showed 99% to 100 % nucleotide identity to those of Clonostachys rosea (NCBI accession Nos. MZ424803-MZ424809 (ITS), and MZ451383-MZ451389 (TEF1-α)). BLAST analysis of the ITS and TEF1-α sequences of isolates, DG-3, DG-3-1, and DG-4 in the NCBI database produced matches of 99.1% or better to Fusarium acuminatum (NCBI accession Nos. MZ424810-MZ424812 (ITS), and MZ441148-MZ441150 (TEF1-α)). Conidia of C. rosea were hyaline, round or oval, aseptate, with an average length and width of 6.5-8.5×2.5-3.5 μm. Chlamydospores were round or oval, with an average length and width of 9.9-16.2×8.1-13.6 μm. Conidiophores of C. rosea were hyaline, septate, Verticillium-like, conidiogenous cells were slender and cylindrical. Macroconidia of F. acuminatum were hyaline, falciform, 0-5 septate, with an average length and width of 22.5-39.5 × 2.5-4.5 μm. Microconidia were rare. Chlamydospores were oval, with an average length and width of 9.5-14.3×8.1-11.8 μm. Conidiophores of F. acuminatum were hyaline, septate, branched (Fig. S2). A pathogenicity test was performed by inoculating a conidial suspension of 1×105 per ml in 0.025% Tween 20 onto roots of A. sinensis. Inoculated roots were incubated in a humid and dark chamber at 26°C for 24 h, and then with 12 h light/12 h dark for six days. The pathogenicity assay was repeated three times for each isolate. Root rot was observed seven days after inoculation and mycelia were observed on the surface of the lesions (Fig. S1). Two isolates were recovered from the lesions and named DG-2-D and DG-3-1-D. The colonial morphology of these two isolates and the original isolates DG-2 and DG-3-1 was identical after six days of inoculation on PDA (potato dextrose agar) (Fig. S2). The two isolates recovered were identified as C. rosea and F. acuminatum, respectively, by amplifying and sequencing a portion of the TEF1-α gene. To our knowledge, this is the first report of C. rosea causing root rot of A. sinensis in China. Fusarium acuminatum was reported to cause root rot of A. sinensis in Gansu province, China in 2021 (Niu et al. 2021). Clonostachys rosea has been previously reported to cause root rot of soybean in the United States (Bienapfl et al. 2012). The disease must be considered in existing management practices, and our findings provide a foundation for management this disease.

First Report of Collar Canker and Dieback of Camellia sinensis Caused by Fusarium solani Species Complex in Henan, China.

Tea (Camellia sinensis) is a significant commercial crop cultivated in China. A collar canker and dieback disease of tea has recently been reported in Sri Lanka (Sinniah et al. 2017). During the Spring of 2020, tea bushes with symptoms of fewer leaves, yellowing of leaves with cankers having cracks and peeling of bark, premature falling in the early stage and progressive dieback of branches followed by death of the whole tree that were partially covered with white-colored mold were observed in tea plantations over a 200 hm2 area in Xinyang, Henan Province, China (Figure 1A). To determine the causative agent, dead tea plant stems with white-colored mold were collected, cut into small pieces and surface-sterilized with 75% ethanol, followed by rinsing twice with sterile distilled water and then air-drying. The pieces were placed on potato dextrose agar (PDA) and incubated at 28℃ for 7 days. A total of 19 fungal colonies were isolated and transferred to fresh PDA. Single-spore isolates were made for each and all were observed to produce white flocculent mycelium with the reverse side being pale yellow on PDA. The appearance of a representative strain, XYWLD-07, on PDA is shown (Figure 1B and 1C). Macroconidia (14.26 to 42.13 × 3.53 to 6.79μm) and microconidia (6.93 to 11.85 × 2.72 to 6.33μm) were observed in the cultures (Figure 1E). The genomic DNA from XYWLD-07 was extracted and the internal transcribed spacer region (ITS) of ribosomal DNA, beta-tubulin, translation elongation factor 1-alpha (TEF- 1α), RNA polymerase I beta subunit (RPB1) and RNA polymerase II beta subunit (RPB2) genes were amplified using the primer pairs ITS1/ITS4 (5'- CTTGGTCATTTAGAGGAAGTAA -3'/5'- TCCTCCGCTTATTGATATGC -3') (Gardes & Bruns 1993) , Bt-F/Bt-R (5'- AACATGCGTGAGATTGTAAGT -3'/5'- TCTGGATGTTGTTGGGAATCC -3'), EF-1/EF-2 (5'- ATGGGTAAGGARGACAAGAC -3'/5'- GGARGTACCAGTSATCATGTT -3'), RPB-1F/1R (5'- TTTTCCTCACAAAGGAGCAAATCATG -3'/5'- GTTCACCCAAGATATGGTCGAAAGCC -3') and RPB2-5F2/11aR (5'- GGGGWGAYCAGAAGAAGGC -3'/5'- GCRTGGATCTTRTCRTCSACC -3') (O'Donnell et al. 2010), respectively. The PCR products were sequenced and deposited in GenBank (Accession Nos. MZ411431, MZ439920, OL982608, OM069738 and OM265212), respectively. BLASTn against NCBI nr and Fusarium-ID database revealed that the ITS sequence had 99.65% homology with BLAST sequences of F. solani (GenBank Accession Nos. JN882257.1 and JN882255.1), the beta tubulin sequence had 96.92% similarity with BLAST sequences of F. solani (KU983876.1 and MN692929.1), TEF- 1α gene sequence had 99.15% and 99.29% homology with BLAST sequences of F. solani (KM096405.1 and KM096409.1), RPB1 gene sequence had 99.32% and 99.41% homology with BLAST sequences of F. solani (KU974331.1 and MT305119.1) and RPB2 gene sequence had 98.15% and 98.20% homology with BLAST sequences of F. solani (MT305182.1 and MT305177.1), respectively. Furthermore, TEF- 1α, RPB1 and RPB2 gene sequences had more than 98.52% match with F. solani sequences in Fusarium-ID databases. To test for pathogenicity, the inoculum was grown for 10 days at 25°C on 200g sterilized boiled wheat grain and sand (3:1, v/v) with 20-ml spore suspension (107 conidia /ml) of strain XYWLD-07. Two-year tea seedlings were planted in pots of autoclaved soil with 5% (m/m) strain XYWLD-07 inoculum but controls were grown in autoclaved soil with only sterile wheat/sand mix. The experiment was performed five times. After 30 days, collar canker symptoms appeared on inoculated tea branches, whereas the control was asymptomatic (Figure 1F). The fungal pathogen was reisolated from the symptomatic tea tissues and again identified as F. solani based on the above analysis of the five gene sequences (Figure 1D). F. solani has been previously identified as causal agent of collar canker and dieback of C. sinensis in Sri Lanka (Sinniah et al. 2017) and India (Sarmah et al. 2017).To our knowledge, this is the first report of F. solani causing collar canker and dieback of C. sinensis in China. As this disease could result in death of tea bushes, effective management strategies should be investigated and applied.

Establishment and application of a qPCR diagnostic method for Theileria annulata.

Bovine theileriosis caused by several Theileria species including Theileria annulata, Theileria parva, Theileria orientalis, Theileria mutans, and Theileria sinensis is a significant hemoprotozoan tick-borne disease. Among these, Theileria species, T. annulata, which causes tropical theileriosis (TT), is regarded as one of the most pathogenic and is responsible for high mortality. At present, most conventional diagnostic methods for tropical theileriosis are time-consuming and laborious and cannot distinguish newfound T. sinensis in China. Therefore, a high sensitivity and specificity real-time quantitative PCR method based on the TA19140 target molecule was developed, and the method was found to be specific for T. annulata. No cross-reaction was observed with T. sinensis, T. orientalis, Babesia bovis, Babesia bigemina, or Hyalomma anatolicum which is negative for T. annulata. A total of 809 field samples from different regions of China were analyzed by using the developed qPCR and conventional PCR. The positive samples for T. annulata detected by real-time qPCR and conventional PCR were 66/809 (8.16%) and 20/809 (2.47%), respectively, and all positive amplicons by qPCR were confirmed by Sanger sequencing. The results showed that the developed qPCR for the T. annulata 19,140 gene was more sensitive than conventional PCR. In addition, we first discovered that TA19140 was mainly expressed at the schizont and merozoite stages of T. annulata by relative quantification. The protein encoded by the TA19140 gene may be used as a potential diagnostic antigen for tropical theileriosis. In conclusion, a real-time quantitative PCR diagnostic method targeting the TA19140 gene was successfully established and could be used for both the quantitative and qualitative analysis of T. annulata infection from cattle and vector ticks, which will greatly help to control and diagnosis of tropical theileriosis.

基于MaxEnt模型的中国沙棘潜在适宜分布区分析

PDF HTML阅读 XML下载 导出引用 引用提醒 基于MaxEnt模型的中国沙棘潜在适宜分布区分析 DOI: 10.5846/stxb202101260269 作者: 作者单位: 作者简介: 通讯作者: 中图分类号: 基金项目: 科技部中俄科技合作专项（2014DFR31230）；甘肃省自然科学基金（18JR3RA177）；"中-俄沙棘加工技术甘肃省国际科技合作基地"项目（GHJD201802）资助 Identification of potential distribution area for Hippophae rhamnoides subsp. sinensis by the MaxEnt model Author: Affiliation: Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:中国沙棘主要分布于我国华北、西北、西南等地森林-草原过渡地带，是我国北方地区退耕还林、生态修复等工程的重要造林树种，对维持干旱、半干旱地区的生态环境稳定具有重要意义。探讨限制中国沙棘分布的主导气候因子，模拟其潜在适宜分布区，以期为中国沙棘在林业生态工程和生态经济林建设中的合理种植和推广提供理论依据。基于中国沙棘自然分布的328个地理样点，利用最大熵（MaxEnt）模型对中国沙棘的潜在分布区的主导气候因子进行分析，并预测中国沙棘的潜在分布范围。结果表明，基于气候变量的MaxEnt模型训练集和测试集受试者工作特征曲线下面积（AUC值）分别为0.962±0.001和0.949±0.001，均大于0.9，表明MaxEnt模型对中国沙棘潜在分布区的预测具有极高的准确度，可信度好。基于环境变量贡献率和刀切法的结果表明年降雨情况、生长季的水热状况、最干季降雨和最冷月最低温等是限制中国沙棘分布的主要气候因素，其中年降雨是限制中国沙棘分布的主导气候因子。通过模拟得到现代中国沙棘潜在地理分布的总适生区面积为165.1万km2；其中高适生区和中适生区面积共93.3万km2，主要集中分布于河北西部、北部，山西全境，陕西北部及秦岭山区，宁夏南部，甘肃东部、南部及祁连山区，青海东部，四川西部和西藏东部及中部地区；在吉林东北部，内蒙古中部、南部，以及河南西北部地区也有少量分布。 Abstract:Hippophae rhamnoides subsp. sinensis is mainly distributed in the forest-grass transition zone and is an important afforestation tree species for the conversion of farmland to forests and ecological restoration projects in North, Northwest, and Southwest China, which plays an important role in maintaining the stability of the ecological environment. This paper tries to explore the dominant climatic factors that limit the distribution of H. rhamnoides subsp. sinensis in China, and to simulate its suitable distribution area. In order to provide a theoretical basis for the overall arrangement and promotion of H. rhamnoides subsp. sinensis in forestry ecological engineering and ecological economic forest construction. Based on 328 geographical distribution samples within the distribution range of H. rhamnoides subsp. sinensis, the maximum entropy (MaxEnt) model is used to predict the potential distribution range of H. rhamnoides subsp. sinensis in China. The receiver operating characteristic curve, Jackknife test and the precent contribution of the climatic factors are carried out to analyze to the climatic factors limiting the modern potential geographical distribution of H. rhamnoides subsp. sinensis, and the response curve is used to determine the suitable range of climatic factors. The results showed that the average values of the area under the receiver operating characteristic curve (AUC) of training and test data were 0.962±0.001 and 0.949±0.001 respectively, higher than 0.9, indicated the accuracy of the MaxEnt model was pretty high for modeling potential distribution regions of H. rhamnoides subsp. sinensis. The combined results from Jackknife test and the precent contribution of the climatic factors revealed that annual precipitation, temperature and precipitation of growth season, precipitation of driest quarter and min temperature of coldest month were the key climate factors that resctrited the distribution of H. rhamnoides subsp. sinensis in China. The total suitable area of the modern potentially geographical distribution of H. rhamnoides subsp. sinensis based on the climatic factors was 165.1×104 km2. And the total area of the potential most suitable region and suitable region of H. rhamnoides subsp. sinensis in China was 93.3×104 km2, mainly located in western and northern Hebei, Shanxi, northern Shaanxi and Qinling Mountains, southern Ningxia, eastern and southern Gansu, Qilian Mountains, eastern Qinghai, western Sichuan, eastern and central Tibet, moreover, there were few suitable region were located in northeastern Jilin, the central and southern Inner Mongolia, as well as northwestern Henan. These regions can be used as the dominant areas for the cultivation and prometon of H. rhamnoides subsp. sinensis. 参考文献 相似文献 引证文献

First Report of Pestalotiopsis chamaeropis Causing Leaf Spot on Eurya nitida in China.

Eurya nitida Korth. belonging to the family Theaceae is an evergreen shrub or small tree and is usually used as a very important ornamental tree and nectar source plant (Khan et al. 1992; Ma et al. 2013). It also has high medicinal values with the treatment of rheumatoid arthritis, diarrhea, innominate inflammatory of unknown origin, ulcer fester and traumatic hemorrhage (Park et al. 2004). In October 2020, symptoms of leaf spot were observed on E. nitida in Meiling Scenic Spot of Nanchang, Jiangxi Province, China (28.78°N, 115.83°E). We surveyed about 300 m2 of the mountain area which holds about 100 trees of E. nitida scattered naturally near the waterside or regularly planted on either side of the mountain road. Most of the infected plants were observed from humid environments or waterside, with 15~20% disease incidence, and the disease severity on a plant basis was determined to be 25% to 30%, depending on the field. Sixty infected leaves were collected from 20 individual trees which have the same symptoms. The symptoms on infected leaves appeared as tiny circular spots that gradually enlarged into brown circular necrotic lesions and then became a light gray with brown borders and black acervuli at the later stages of the disease. Ten leaves of infected tissues randomly selected from collected sixty infected leaves were cut into 4 mm2 pieces, and surface disinfected with 75% ethanol for 30s and 1% hypochlorite for 1 min, rinsed three times with sterile water, plated on potato dextrose agar (PDA), and incubated at 25°C in the dark for 5 to 7 days. Five isolates with similar morphological characteristics were obtained. Colonies developed copious white aerial mycelium covering the entire Petri dish area after 7 to 10 days. Conidiogenous cells were discrete, hyaline, and smooth. Conidia were fusiform, ellipsoid, 4-euseptate and ranged from 21.86 to 29.80 × 5.95 to 9.80 µm. Apical cells were hyaline with 2 to 3 unbranched, tubular apical appendages (mostly 3); basal cell was hyaline, obconic with a truncate base; three median cells doliiform to subcylindrical, brown. The morphological characteristics of all isolates matched features described for Pestalotiopsis chamaeropis Maharachch., K.D. Hyde & Crous (Maharachchikumbura et al. 2014). Two single representatives (JAUCC L001-1 and JAUCC L002) were used for molecular identification, which were verified based on the amplification of DNA sequences of internal transcribed spacer region (ITS) gene and translation elongation factor 1 alpha (TEF1-α) gene, using the primers ITS4/ITS5 (White et al. 1990) and EF1-526F/EF1-1567R (Rehner and Buckley 2005), respectively. The sequenced loci (GenBank accession nos. ITS: MW845761, MW828589 and TEF1-α: MW838967, MZ292464) exhibited over 99% homology with P. chamaeropis strain CBS 186.71 in GenBank (GenBank accession nos. KM199326 and KM199473), confirming the morphological identification. Phylogenetic reconstruction was generated by using the maximum likelihood (ML) method based on the Kimura 2-parameter model, with bootstrap nodal support for 1000 pseudoreplicates in MEGA software, version 7.0. The result showed that our isolates were clustered together with P. chamaeropis at 99% bootstrap values. Based on morphological characteristics and molecular phylogenetic analysis, the isolates were identified as P. chamaeropis. The pathogenicity of one representative isolate (JAUCC L001-1) was tested indoor by inoculating the top leaves of six healthy E. nitida plants. Three plants with three leaves were punctured with flamed needles and sprayed with a conidial suspension (1 × 106 conidia/ml), and other three plants wounded inoculated with mycelial plugs (5 × 5 mm3). Mock inoculations were used as controls with sterile water and non-infested PDA plugs on three leaves each. Treated plants were incubated in an artificial climate box with high relative humidity at 25 °C. After 10 days, symptoms on all wounded inoculated plants were similar to those previously observed with distinct tiny circular spots, whereas no symptoms appeared on inoculated plants. Pestalotiopsis chamaeropis was re-isolated from symptomatic tissues but not from the mock-inoculated plants, and its identity was confirmed by morphological characteristics and molecular data, which confirmed Koch's postulates. Pestalotiopsis chamaeropis was previously reported as the causal agent of leaf blight diseases on Camellia sinensis in China (Chen et al. 2020), Pieris japonica in Japan (Nozawa et al. 2019) and Prostanthera rotundifolia in Australia (Azin et al. 2015). To our knowledge, this is the first report of P. chamaeropis causing a leaf spot disease on E. nitida in China, and this disease may be more widespread than the sampled location. This finds is beneficial to the better protection of E. nitida, a widespread medicinal and nectar source plant with high economic value.

First report that Colletotrichum aenigma causes leaf spots on Aquilaria sinensis in China.

Aquilaria sinensis (Lour.) Spreng, also known as eaglewood, belongs to the Thymelaeaceae family and has a considerably high medicinal value. It has been enlisted as the class II national key protective plant. In June 2019, about 15 percent of A. sinensis treelets in a forest area of China's Hainan province were observed to have the anthracnose symptoms. The diseased spots on leaves of A. sinensis treelets were usually round or irregular with pale yellow edges. The color of the center of the lesion was firstly light brown and then black or yellowish-brown. Small pieces of tissue from the edge of the leaf spots were surface sterilized in 75% alcohol for the 60s, washed twice with sterile distilled water, and then cultivated at 28 °C in darkness on potato dextrose agar (PDA) medium. One fungus was systematically isolated to get pure cultures. The culturing of the three isolates was carried out in PDA media at 28 °C for a week. The average diameter of the collateral colony was 6.80 ±0.60 cm. Initially, the fungal colonies were white aerial mycelium and the central area of the colonies slowly turned jacinth. After seven days, the central mycelium turns grayish-green and the colonies' undersurfaces were grey to white. The colony's surfaces were fluffy and round with smooth edges. Conidia were cylindrical, smooth, and transparent, with a slight indentation in the middle and uneven distribution of small particles inside, 12.5-20.6×3.5-6.8 µm (ave=15.9±1.40×5.18±1.07, n=50). Appressoria were typically elliptic or irregular and brown to dark brown. The isolates were characterized as Colletotrichum gloeosporioides species complex on the basis of the conidial morphology and culture representation, (Deng et al. 2017; Weir et al. 2012). To further verify the identification of the species, CX-0301, the isolated representative strains were extracted for genomic DNA. mating type 1-2-1 (Mat-1-2-1) ApMat, actin (ACT) gene, chitin synthase (CHS), and beta-tubulin (TUB2) gene were amplified using the primer pairs VcaMat-5F/VcaMat-5R, ACT-512F/ACT-783R, CHS-1-79F/CHS-1-354R, and TUB2-T1/Bt2b, respectively (Damm et al. 2012; Du et al. 2005). The homologous sequences of MN310694, MN310693, MN310692, and MN310691 were submitted to GenBank. These genes have ≥a 97% sequence similarity to the genes of Colletotrichum aenigma (MG717319.1, MG717317.1, MH476565.1, MH853679.1, respectively) in GenBank. These morphological and molecular characteristics identified that the pathogen is C. aenigma. (Weir et al. 2012). To further verify the isolated pathogen, the pathogenicity test was performed on uninfected healthy 2-year-old eaglewood seedlings. The conidial suspension (1×106 conidia/ml) of 5ml was sprayed on both surfaces of 10 leaves of plants of the same age and height and the controls were treated solely with distilled water (Deng et al. 2017). Upon completion of inoculation, plants were kept under greenhouse conditions with an assigned temperature of 28 ± 2°C while keeping relative humidity to 90% on a 12-h fluorescent light/dark regime. Anthracnose-like symptoms were observed 6 days postinoculation. The control plant tissues remained healthy. Follow up reisolation of C. enigma culture was obtained in PDA agar plates from leaf infected lesions, and the morphological features were found to be consistent with that of CX-0301 isolate, satisfying Koch's postulates. Based on the characterized information, it is the first report of Colletotrichum aenigma responsible for causing leaf spots on Aquilaria sinensis in China. Thereby, this provides a theoretical reference for the research and control of anthracnose on A. sinensis.

First Report of leaf spot disease caused by Colletotrichum gloeosporioides on Chaenomeles sinensis in China.

Chaenomeles sinensis is a shrub or small arbor of the genus Chaenomeles in Rosaceae, which is widely planted in China. It is a kind of garden ornamental plant and has high economic value. Since 2020, a leaf disease occurred on the foliage of C. sinensis at the campus of Nanjing Forestry University, Nanjing, China. After investigating, C. sinensis was found with leaf spot disease at a 100% infection rate, which causing gigantic ornamental loss. Leaf spots are round to irregular distributing on the leaves, in addition, the color of spots is brown. There are yellow halos on the edge of the lesion. Small leaf tissues (3 to 4 mm2) from lesion margins were surface sterilized with 75% ethanol for 30s and then rinsed with sterile dH2O for three times. Afterwards, placed on potato dextrose agar (PDA) at 25°C. Pure cultures were obtained by monosporic isolation, and a representative isolate (NJTJ.1) was obtained. When cultured on PDA, the colony of NJTJ.1 was white and cottony. On the reverse side, the color of colony nearly light yellow. The colony were placed in the liquid Carboxymethyl cellulose (CMC) medium. After culturing for 24h in a shaker at 25℃ and 150rmp/min, the spore liquid was taken by us. The conidia were one-celled, straight, hyaline, subcylindrical with rounded ends and measured 15.1 to 23.6× 5.4 to 7.9 µm (n =30). Appressoria were one-celled, brown, thick-walled, ellipsoidal, and measured 7.7 to 13.8 × 6.4 to 10.3 µm (n =30). The morphological characteristics of NJTJ.1 fitted with the description of the Colletotrichhum gloeosporioides complex (Weir et al., 2012). For accurate identification, the internal transcribed spacer (ITS), and the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT) and chitin synthase (CHS-1) were amplified with primers ITS1/ITS4, GDF/GDR, ACT-512F/ACT-783R, and CHS-79F/CHS-345R (Zhu et al, 2019), respectively. The sequences were deposited in GenBank [Accession Nos.MT984264, MW030495 and MW030496 to MW030497 for NJTJ.1]. A Blast search of GenBank showed that ITS, GAPDH, ACT and CHS-1 sequences of NJTJ.1 were 99%, 99%, 100% and 100% identical to those of C. gloeosporioides (MH571757.1 ,KY995355.1 , MN058143.1 and MN313581.1). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate NJTJ.1 clustered in the C. gloeosporioides clade with 99% bootstrap support. The pathogenicity of the NJTJ.1 was verified both on detached and living leaves. The detached leaves were inoculated with 5-mm mycelial plugs cut from the edge of 6-day old cultures on PDA and 20 μL of spore suspension (106 conidia/mL) and each treatment had 5 replicates. Controls were treated with sterile dH2O. The inocula were placed at a distance of 2 to 3 cm on the leaves which were wounded with a sterile needle. All of them were placed in 20-cm dishes on wet filter paper at 25°C. After 5 days, all the inoculated points showed lesions which were similar to those outdoor observed. Whereas, controls were asymptomatic.At the same time, the plugs of C. gloeosporioides were inoculated on living leaves.After 7 days, the leaves which were inoculated also appeared lesions. This is the first report of C. gloeosporioides causing leaf blotch on Chaenomeles sinensis in China. These data will help develop effective strategies for managing this disease.