Genomic Characterization and Molecular Detection of Rehmannia Allexivirus Virus, a Novel Allexivirus Infecting Rehmannia glutinosa.

Rehmannia glutinosa is one of the most important medicinal plants in China and is affected by viral diseases. In this study, a new virus tentatively named Rehmannia Allexivirus virus (ReAV) was identified through high-throughput sequencing, reverse-transcription polymerase chain reaction (RT-PCR), and Sanger sequencing. The complete genome length was 7297 nt and it contained five open reading frames (ORFs) encoding replicase, triple gene block 1(TGB1), TGB2, TGB3, and coat protein (CP). The replicase and CP presented nucleotide homology ranges of 59.9-65.2% and 47.5-55.5% between the nine ReAV isolates and the other 12 species of the genus Allexivirus. In the nine isolates, ReAV-20 and ReAV-31 isolates showed breakpoints in the replicase and CP regions, respectively. The other isolates shared 87.2-96.5% nt with the whole genome nucleotide identity. The phylogenetic tree showed that seven ReAV isolates based on replicase, CP, and whole genome sequences were clustered in the same branch and were related to the genus Allexivirus. The ReAV detection rates for 60 R. glutinosa samples were 73.3-81.7% through RT-PCR using primers targeting the replicase or CP genes. These results demonstrate that ReAV is the dominant virus in R. glutinosa. This study provides important evidence for understanding viruses infecting R. glutinosa and for establishing efficient strategies to prevent viral spread.

First Report of Aspergillus westerdijkiae Causing Leaf Blight on Rehmannia glutinosa in China.

Rehmannia glutinosa (also known as Chinese foxglove) is a perennial dicotyledonous herb, which plays an important role in traditional Chinese medicine. Its active ingredients have a wide range of pharmacological effects on the blood system, endocrine system, immune system, cardiovascular system, and nervous system (Zhang et al. 2008). In May 2022, leaf blight was observed on 45-day-old R. glutinosa in a seedling nursery in Jiaozuo City (35°01'44.20″N, 113°05'30.63″E), Henan Province, China with an approximate disease incidence up to 54% (~1,300 plants). Irregular brown lesion initially appeared on the tips of basal leaves, then progressed to the entire leaf causing leaf drying out (Supple. Fig. 1-A, B, C). The same symptoms appeared successively in the leaves from the base to the top of the plant, which eventually caused the whole plant to die. To identify the pathogen, eight symptomatic leaves were randomly collected from eight individual plants, and cut into small pieces (5 × 5 mm) at the border of lesions. The pieces were surface disinfected in 75% ethanol for 15 s, followed by 1% NaClO for 1 min, rinsed in sterile water three times, and placed on potato dextrose agar (PDA) medium in the dark for 3 days at 25℃. Finally, 12 purified isolates (DHY1-DHY12) were obtained by using single spore method. Leaves of R. glutinosa seedlings were inoculated with conidial suspension (106 conidia/ml), three plants were inoculated per isolate. Controls were treated with sterilized water. All inoculated and control plants were incubated in a greenhouse at 25℃ under 80 ± 10% humidity and a 8-h/16-h dark/light cycle. This experiment was repeated three times. After 5 days, similar symptoms to those of diseased leaves in the seedling nursery appeared on leaves inoculated with DHY4-DHY10, while plants inoculated with DHY1-DHY3, DHY11-DHY12, and the controls remained asymptomatic (Supple. Fig.1-D, E). The same fungi were re-isolated from diseased leaves, fulfilling Koch's postulates. The causal agents DHY4 to DHY10, showed similar morphology, which were morphologically identified as Aspergillus sp. (Visagie et al. 2014). Isolate DHY5 was selected for further study. On PDA plates, the colonies were covered with white velutinous mycelia (Supple. Fig.1-F). Conidia were ochre yellow and outwards concentric circles. Vesicles were globose, and about 20.1-26.6 µm in diameter (Supple. Fig.1-G). Conidiophore stipes were smooth walled and hyaline, with conidial heads radiating. The conidia were light yellow to orange, exudate clear to orange droplets. The conidia were (2.53-3.25) µm × (2.58-3.47) µm in diameter (n=50) (Supple. Fig.1-H). For further molecular identification, the ITS and TUB gene sequences were amplified with primer pairs ITS1/ITS4 and BT2a/BT2b (Glass and Donaldson. 1995), respectively. BLASTn searches of the ITS (PP355445) and TUB (PP382788) sequences showed 100% and 98.42% similarity to those of A. westerdijkiae (OP237108 and OP700424), respectively. Phylogenetic analysis based on the concatenated sequences of ITS and TUB confirmed that the fungus was A. westerdijkiae, (Supple. Fig.2). A. westerdijkiae was mainly reported on its secondary metabolite ochratoxin A contamination of agricultural products, fruits, and various food products, such as coffee beans (Alvindia et al 2016), grapes (Díaz et al. 2009), oranges and fruit juice (Marino et al. 2009), etc. To our knowledge, this is the first report of A. westerdijkiae causing leaf blight on R. glutinosa in China.

First report of basal stem rot caused by three Fusarium species on Chinese yam in China.

The cultivated variety of Chinese yam (Dioscorea polystachya Turcz. cv. Tiegun) is an economically important plant, capable of producing tubers that are used as food and traditional Chinese medicine. The basal stem rot was found on approximately 65% of yam (tuber expansion stage) in a total of 10 ha field in Wuzhi, Wen, and Hua counties, Henan, China (Sep 2021). Dark brown fusiform lesions initially occurred at the stems basal, irregularly extending to join together and leading to loop-stem necrotic indentation. Three diseased samples from Wuzhi county were collected, cut into 5 × 5 mm pieces, surface sterilized in 75% ethanol (30 s) and 1% NaClO (1 min), washed in sterile water 3 times, and placed on PDA in the dark for 3 days at 28℃. A total of 44 isolates forming three groups of Fusarium colonies were obtained using monosporic isolation, of which 19, 8, and 17 isolates were identified as F. oxysporum, F. solani, and F. proliferatum based on colony morphology, respectively. Typical isolates SYJJ6, 9, and 10 for each group were further studied. The SYJJ6 colonies showed gray white abundant fluffy aerial mycelium with rough edges, formation of ellipsoid, unicellular microconidia without septa, 5.6 to 13.4 × 2.4 to 4.7 µm (n = 50), and sickle-shaped, slightly curved macroconidia with 2 to 4 septa, 14.0 to 23.9 × 3.4 to 5.1 µm (n = 50). Isolate SYJJ9 produced flocculent white colonies, grew in a circular pattern with a sharp edge, forming oval or oblong microconidia with zero or one septum, 11.2 to 18.8 × 3.4 to 6.2 µm (n = 50), and slightly curved macroconidia with 2 to 3 septa, 27.6 to 44.0 × 3.9 to 7.4 µm (n = 50). SYJJ10 produced whitish or pinkish white colonies with fluffy aerial mycelium and a red pigmentation, produced renal or oval microconidia with no septa, 5.1 to 11.8 × 1.8 to 4.2 µm (n = 50), and falcate, slightly curved macroconidia with 3 to 4 septa, 16.1 to 30.2 × 3.1 to 5.9 µm (n = 50). Additionally, TUB, EF-1α, and RPB2 genes were amplified with primers BT2a/BT2b, EF1/EF2, and 5f2/-7cr, respectively (Glass and Donaldson 1995; O'Donnell et al. 1998, 2010). BLASTn analysis on SYJJ6 (OR047663, OR047666, OR047669), SYJJ9 (OR047665, OR047667, OR047670), and SYJJ10 (OR047664, OR047668, OR047671) gene sequences were over 99% identical to those of F. oxysporum (100%, MK432917; 100%, MN417196; 99.61%, MN457531), F. solani (100%, MF662662; 100%, MN223440; 99.80%, CP104055), and F. proliferatum (100%, ON557521; 100%, ON458137; 99.90%, LT841266), respectively. Pathogenicity tests of three isolates were separately performed on 60-day-old yam seedlings. The basal stems were wounded using needle, and the wounds were wrapped with cotton balls soaked with conidial suspension (1 mL, 3×106 conidia/mL) or water (control). Each isolate treated three plants and repeated three times. All plants were grown at 28℃ under a 16/8-h light/dark cycle. Typical symptoms emerged on basal stems at 16, 13, and 17 days after inoculation with the conidia of isolates SYJJ6, 9, and 10, while the control basal stems appeared healthy. The re-isolated fungi were identical to the original three isolates. Fusarium species (F. oxysporum, F. commune, F. humuli, etc.)were previously reported to cause wilt or stem rot on different D. polystachya cultivars (Fang et al. 2020; Li et al. 2023; Zhao et al. 2013), or basal stem rot on Panax ginseng (Ma et al. 2020). This is the first report of Chinese yam basal stem rot caused by Fusarium species, which threatens the production of Chinese yam 'Tiegun' and should be further studied.

First Report of Cercospora Leaf Spot Caused by Cercospora cf. flagellaris on Lonicera japonica Thunb. in Henan, China.

Honeysuckle flower (Lonicera japonica Thunb.) is a traditional Chinese medicinal plant. It is perennial and widely cultivated in China, Japan and Korea. From late August to October in 2021 and 2022, leaf spots symptoms were observed on L. japonica in different planting fields in Yuzhou, Yuanyang and Fenqiu districts, Henan province, China. The disease incidence was above 85% which reduce photosynthesis. Early disease symptoms appeared as small, circular to elliptical, brown spots on the leaves and later the lesions (1 to 5 mm × 1 to 4 mm) slowly developed yellow haloes. The different brown lesions seldom merge and form larger irregular lesions. Small fragments (3 to 5 mm) of leave tissue were excised from the lesion margins and surface-sterilized in 3% NaClO for 3 min, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. A total number of 8 cultures were obtained and purified by single-spore subcultures on PDA for morphological identification. The colonies on PDA were whitish to gray, with cottony aerial mycelium. Conidiophores were fasciculate, olivaceous brown, straight or geniculate, uniform in width, multiseptate, and ranged from 290 to 700 µm (560 µm on average, n = 20). Conidia were hyaline, slightly curved or straight, needle shaped, truncate at the base, and terminal at the tip, 3 to 17-septate, and measuring 150 to 240 µm (180 µm on average, n = 20). The morphological features were consistent with Cercospora cf. flagellaris Ellis & G. Martin (Groenewald et al. 2013). The genomic DNA was extracted using CTAB method. The nuclear ribosomal internal transcribed spacer region (ITS), portions of the actin (ACT), histone H3 (HIS3), and translation elongation factor 1-α (TEF1) genes were amplified using primers ITS1/ITS4 (Groenewald et al. 2013), ACT-512F/ACT-783R (Carbone and Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2006), and EF1-728F/EF1-986R (Carbone and Kohn 1999). The resulting 537-bp ITS, 226-bp ACT, 410-bp HIS3, and 306-bp TEF1 sequences of isolate JDJ002 were deposited in GenBank (accession nos. OR492367, OR548247, OR548248 and OR548248, respectively). Sequence analysis revealed that ITS, ACT, HIS3 and TEF1α sequences exhibited ≥99% of identity with the ITS (KP896013), ACT(KP895965), HIS3(MK991295) and TEF1 (MN180408) sequences of C. cf. flagellaris, respectively. A pathogenicity test was conducted on healthy of L. japonica leaves. The healthy leaves pricked from L. japonica plants, rinsed in autoclaved distilled water three times and dried with distilled filter paper. Then twelve healthy leave were inoculated with a mycelial plug (0.4 cm diameter) harvested from the periphery of two week-old colony. As negative control, leaves inoculated with PDA medium plugs. Inoculated leaves were covered with plastic bags to maintain high relative humidity and incubated at 25°C in growth chamber. After 7 days, the inoculated leaves showed symptoms identical to those observed in the field under natural conditions, whereas negative control remained symptom-free. Re-isolation of the fungus from lesions on inoculated leaves confirmed that the causal agent was C. cf. flagellaris. Pathogenicity tests were repeated three times by the same methods with the same results. To our knowledge, this is the first report of C. cf. flagellaris except Cercospora rhamni Fack., Alternaria alternata, Corynespora cassiicola or Phomopsis sp. causing leave spots on L. japonica in China.

Facultative symbionts are potential agents of symbiont-mediated RNAi in aphids.

Aphids are major crop pests, and they can be controlled through the application of the promising RNA interference (RNAi) techniques. However, chemical synthesis yield of dsRNA for RNAi is low and costly. Another sustainable aphid pest control strategy takes advantage of symbiont-mediated RNAi (SMR), which can generate dsRNA by engineered microbes. Aphid host the obligate endosymbiont Buchnera aphidicola and various facultative symbionts that not only have a wide host range but are also vertically and horizontally transmitted. Thus, we described the potential of facultative symbionts in aphid pest control by SMR. We summarized the community and host range of these facultative symbionts, and then reviewed their probable horizontal transmitted routes and ecological functions. Moreover, recent advances in the cultivation and genetic engineering of aphid facultative symbionts were discussed. In addition, current legislation of dsRNA-based pest control strategies and their safety assessments were reviewed.

First Report of Youcai Mosaic Virus Infecting Yam in China.

Yam (Dioscorea opposita Thunb.) is cultivated mainly as a functional food and for nutritional and medicinal purposes in China (1). It is propagated through tubers and this facilitates the spread and accumulation of viruses in the crop, eventually leading to yield losses (2). At present, different virus species belonging to the genera Aureusvirus, Badnavirus, Carlavirus, Comovirus, Cucumovirus, Fabavirus, Macluravirus, Potexvirus and Potyvirus have been reported in yams (3) and fifteen viruses in these genera have been detected in China. In July 2020, a survey of viral diseases on yam was conducted in plantations of Wenxian and Mengzhou counties in Henan Province, China. Fifty-four leaf samples of Dioscorea opposite showing mosaic and leaf discoloration (Supplementary Fig1) were collected from eight fields (five to ten plants per field). These leaf samples were ground in liquid nitrogen and total RNA was extracted from a portion of the mixed powder using RNAprep Pure Plant Plus Kit (TIANGEN Biotech, Beijing, China). A cDNA library was constructed using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) after ribosomal RNA depletion using Ribo-off rRNA Depletion Kit (Vazyme Biotech, Nanjing, China), and sequenced on the Illumina NovaSeq 6000 system at the Berry Genomics Corporation (Beijing, China). A total of 87,075 contigs (>200 bp) were generated from de novo assembly (CLC Genomic Workbench 10.0) from a total of 34,656,172 paired-end reads. After BLASTn analysis, three contigs with the length of 1009, 1340 and 1859 nucleotides shared 96.33%, 96.72% and 96.29% nt identity respectively with youcai mosaic virus SX isolate, a tobamovirus (YoMV GenBank accession no. JX422022). In addition to YoMV, broad bean wild virus 2 and yam latent virus were also identified, which had previously been reported in yams in China. To confirm the NGS result, total RNAs were extracted from fifty-four above-mentioned samples and RT-PCR was carried out to amplify a 528 bp fragment of the coat protein (CP) of YoMV by using a pair of specific primers CP gene. PCR products with expected size were obtained from 26 out of 54 samples, and seventeen amplicons of YoMV-CP were sequenced (accession nos. ON052726 to ON052742). The nt sequence identities of CP gene among these seventeen isolates were 99.6%-100%. Furthermore, the near-full-length genomic sequence of YoMV-Do41 isolate was obtained from sample 41 by RT-PCR amplification of four overlapping fragments using the following primer pairs: YoMV-15F/YoMV-1910R, YoMV-1770F/YoMV-3750R, YoMV-3645F/YoMV-5404R and YoMV-4921F/YoMV-6280R (Supplementary Table1). The YoMV-Do41 isolate was 6, 274 nt in length (accession no. ON149803) and shared 89.65% and 97.31% nt identities to As1-2 isolate (GenBank accession no. MW307290) and to SX isolate (accession no. JX422022), respectively.To the best of our knowledge, this is the first report of YoMV infecting yam in China. YoMV has a wide host range including genera Impatiens, Rehmannia, Brassica, Chelidonium, Trifolium, Crossandro, Alstroemeria, Stellaria. This study will serve as an important reference for the host range of YoMV. According to the detection rate infections with YoMV in yam are common in these producing regions. Further studies will be required to determine the infection rate in other producing regions and the potential threat posed by YoMV on yam production should be considered.

First Report of Tobacco Mild Green Mosaic Virus Infecting Rehmannia glutinosa in China.

Rehmannia glutinosa (family Scrophulariaceae) is an important traditional medicinal plant, whose root is used to treat anemia, hemoptysis, and gynecological diseases in China (Matsumoto et al. 1989). This plant is native to China and cultivated in China, Korea, Japan, and northern Vietnam (Kwak et al. 2020). Viral diseases caused remarkable loss in the yield and quality of R. glutinosa (Ling et al. 2009). To date, ten viruses have been identified globally to infect R. glutinosa and seven of these viruses reported in China (Liu et al. 2018; Zhang et al. 2021). Most plants of R. glutinosa are infected with one or more of these viruses (Kwak et al. 2018; Zhang et al. 2004). In July 2020, a survey of the viral disease infecting R. glutinosa was conducted in commercial plantations of Wenxian, Wuzhi, Mengzhou, and Yuzhou counties in Henan Province, China. The disease symptoms included mosaic, chlorosis, leaf distortion, and the percentage of symptomatic plants was over 70% in the surveyed fields (n=9). Sixty leaf samples of symptomatic R. glutinosa plants were collected from nine cultivation fields in Wenxian, Wuzhi, Mengzhou, and Yuzhou counties (five to seven plants for each field). Total RNA was extracted from one pooled sample containing a portion of all above-mentioned leaf samples using RNAprep Pure Plant Plus Kit (TIANGEN Biotech, Beijing, China) and analyzed by high-throughput sequencing (HTS) to identify viral pathogens. A transcriptome library was generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA), and sequenced on an Illumina NovaSeq6000 sequencing system at Berry Genomics Corporation (Beijing, China). A total of 27,664,949 high-quality clean reads were obtained after trimming and used for contig assembly. The assembled contigs (n=109,180) were searched using Basic Local Alignment Search Tool (BLAST) at GenBank. BLASTn analysis showed that the R. glutinosa plants were infected with known viruses, including broad bean wilt virus, rehmannia mosaic virus, youcai mosaic virus, and cucurbit chlorotic yellows virus. In addition, one contig (6,418 nt in length) had a nucleotide sequence identity of 99.64% with the TN29 isolate of tobacco mild green mosaic virus (TMGMV, GenBank accession no. MF139550). To confirm the presence of this virus, sixty above-mentioned samples were screened by reverse transcription-polymerase chain reaction (RT-PCR) using the specific primer pairs (Supplementary Table1) TMGMG-CPF/TMGMG-CPR targeting a 545-nt fragment within the CP gene. Amplicons with expected sizes were detected from 47 of 60 samples but not from the negative control (virus-free healthy plant through the tip meristem culture). Seventeen amplicons (11#, 13#, 14#, 21#, 22#, 23#, 25#, 26#, 27#, 31#, 32#, 33#, 37#, 52#, 57#, 59#, and 60#) of TMGMV-CP were selected, and purified. The PCR products were cloned into the pMD19-T vector (TAKARA Biotech, Dalian, China) and sequenced. The sequences were deposited into the GenBank (accession nos. MZ395944 to MZ395960). The near-full-length genomic sequence of TMGMV-Rg14 isolate was obtained from one positive sample (sample no. 14) by RT-PCR amplification of two overlapping fragments using the following primer pairs: TMGMV-40F/TMGMV-3570R and TMGMV-3220F/TMGMV-6400R. The near-full-length genomic sequence of the TMGMV-Rg14 isolate was 6 304 nucleotides (nt) in length and deposited into GenBank (accession no. MZ395975). BLASTn analysis demonstrated that the TMGMV-Rg14 isolate shared a sequence identity ranging from 96.89% (AB078435) to 99.60% (MF139550) with the other TMGMV isolates. Furthermore, the virus-free healthy R. glutinosa plants were inoculated with sap from the positive sample (14#) to confirm the infection of TMGMV. Mosaic symptoms were induced on the systemically infected leaves of the inoculated plants 14 days post inoculation. The systemically infected leaves of inoculated plants were assayed by RT-PCR using the primer pairs TMGMV-CPF/CPR. Amplicons of expected size were detected from the inoculated plants but not from non-inoculated plants. To our knowledge, this is the first report of TMGMV infection on R. glutinosa. Further studies are necessary to select a suitable indicator plant for this TMGMV, its host range, and the symptoms it induces in single infection. Since R. glutinosa is cultivated by vegetative propagation, production of virus-free healthy plants is necessary. This study will help to generate virus-free healthy plants and prevent viral disease on R. glutinosa. Further study is needed to determine its pathological implications and economic impact on R. glutinosa in China.

First Report of Dasheen Mosaic Virus Infecting Typhonium giganteum Engl. (Baifuzi) in Henan Province of China.

Typhonium giganteum Engl. (Baifuzi ) is a perennial plant of the family Araceae. In China, its root is commonly used as an antispasmodic for stroke and cancer treatment (Chi et al. 2010; Gao et al. 2014; Khalivulla et al. 2019). Yuzhou city in Henan Province is the main producing area of T. giganteum Engl., and in July 2020, a survey of viral disease infecting T. giganteum Engl. was conducted in the city. In the surveyed fields (n =5), over 60% of plants displayed varying levels of virus-like symptoms, including mosaic, chlorotic and leaf distortion (Supplementary Figure S1) . To identify possible viral pathogens associated with the disease symptoms afflicting T. giganteum Engl., one leaf each from 25 symptomatic plants was collected and analyzed by high-throughput sequencing (HTS) as well as PCR. For HTS analysis, total RNA was extracted from one pooled sample containing a portion of all abovementioned leaves using RNAprep Pure Plant Plus Kit (TIANGEN Biotech, Beijing, China). After removing ribosomal RNA with Ribo-off rRNA depletion kit (Vazyme Biotech, Nanjing, China), a sequencing library was generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) and sequenced on an Illumina Novaseq6000 sequencing system at Berry Genomics Corporation (Beijing, China). A total of 6,899,143 high-quality clean reads were obtained after trimming and used for contig assembly. BLASTn and BLASTx analyses on the contigs (n = 128,400) showed that one contig (9,245 bp in length) exhibited a sequence identity of 84.0% with the reference sequence of dasheen mosaic virus (DsMV, NCBI reference seq. NC_003537, genus Potyvirus, family Potyviridae) , suggesting infection of the plants by DsMV. No other viral sequences were detected in the sample. To confirm these results, a near full-length genomic sequence of DsMV was obtained from one sample (sample no. 39) by reverse transcription polymerase chain reaction (RT-PCR) of three overlapping fragments with the following primer pairs: DsMV-1F (5'-AAATTAAAACATCTCAACAAAACCTACA-3') /DsMV-4130R (5'-TTCATGGTCCTCGTGGAGTATA-3'), DsMV-3870F (5'-GAGGACGTGAGAATTCAAAGTCT-3')/DsMV-8250R (5'-GTCCAACCTTGCTTGATGCATGC-3'), DsMV-7690F (5'-GGAGCGACTCCTCTTCCAAAGTTGTG-3')/DsMV-10100R (5'-TGAACACCGTGCACGAAGCATCTC-3'). The PCR products were cloned into pMD19-T vector (TAKARA Biotech, Dalian, China) and sequenced. The near full-length genomic sequence of the isolate (DsMV-BF39) was 9,737 nt in length and deposited into GenBank under the accession no. MZ043618. BLASTn analysis of this sequence demonstrated that it shared an identity ranging from 78.6% (MG602234) to 85.6% (MG602227) with various DsMV isolates. To determine whether DsMV was closely associated with the symptoms observed in T. giganteum Engl., leaf tissues from 30 symptomatic plants and 22 asymptomatic plants were analyzed by RT-PCR using primer pairs DsMV-CPF (5'-TGTTCTGTGAACATGATGAAGTTG-3', sense) and DsMV-CPR (5'-GTAACTGTGGCCTGTTTACCAG-3', antisense) targeting a 916 bp fragment of the CP gene of DsMV. Amplicons with the expected size were detected from the 30 symptomatic plants but not from the 22 asymptomatic plants, suggesting a close association between DsMV infection and the observed symptoms. To our knowledge, this is the first report of DsMV infecting T. giganteum Engl.. Further study is needed to identify the specific symptoms induced by this virus in T. giganteum Engl. and to understand the biological characteristics, epidemiology, prevalence of this virus in China.

[Effect of combined application of inorganic and organic fertilizers on growth and quality of Salvia miltiorrhiza].

The study is aimed through field experiments to study the effect of combined application of organic and chemical fertilizers on the growth and quality of Salvia miltiorrhiza, provide ideas for reducing fertilization while increasing the efficiency as well as improving the quality of produces. The experiment included 6 treatments viz., no fertilization(CK), full application of chemical fertilizer(F), 25% orga-nic fertilizer with 75% chemical fertilizer(M25), 50% organic fertilizer with 50% chemical fertilizer(M50), 75% organic fertilizer with 25% chemical fertilizer(M75), and fully apply organic fertilizer(M100). The results showed that:(1)from the perspective of yield and economic benefits, M75 was the best and M100 second;(2)for effective components, the combined application of organic and chemical fertilizers increased the content of main water-soluble components and the total content of effective components, among which M25 and M50 were better.

A Bis(Salamo)-Based Fluorogenic Sensor for Highly Selective and Sequential Recognition of Cu2+ and B4O72- Ions in Semi-Aqueous Medium.

A new type of multifunctional bis(salamo)-based fluorogenic sensor H2BS was designed and synthesized. Under the action of VDMF: VH2O = 9: 1, the fluorogenic sensor can identify Cu2+ and B4O72-, in which N and O atoms can serve as binding sites for Cu2+ and B4O72-, the stoichiometry of the binding of the fluorogenic sensor H2BS and Cu2+ has been confirmed by titration experiment, working curve, ESI-MS analysis and DFT calculation. The pH response experiment also confirmed that the fluorogenic sensor can recognize Cu2+ and B4O72- in the pH range applicable to the physiological environment. The minimum detection limit of H2BS for Cu2+ and B4O72- recognition reaches 1.12 × 10-7 and 5.56 × 10-8 M, and the fluorogenic sensor H2BS has been successfully applied to Cu2+ detection in actual water samples, and the test strip for detecting Cu2+ and B4O72- was obtained. Meanwhile, the success of the test strip experiment made the fluorogenic sensor H2BS to recognize Cu2+ and B4O72- widely used in daily life. A new type of salamo-based multifunctional fluorogenic sensor H2BS was designed and synthesized to identify Cu2+ and B4O72- in aqueous solvent systems. Added Cu2+ to H2BS can cause fluorescence quenching. Further experiments showed that H2BS and Cu2+ form a stable 1:2 complex, while B4O72- can also cause fluorescence quenching of H2BS, which is the occurrence of the PET effect. Meanwhile, H2BS can be used for quantitative detection in the environment and rapid identification in life.

First report of Fusarium proliferatum causing root rot disease in Salvia miltiorrhizae in China.

Salvia miltiorrhiza Bunge is a herb plant used as a traditional Chinese medicine to cure cardiovascular disease. In December 2018, a root rot disease was observed on S. miltiorrhiza in four surveyed counties (Song, Yuzhou, Fangcheng, and Mianchi) in Henan province in China. The disease incidence ranged from 15 to 50% in 12 surveyed fields. At the early stage, the diseased plants were wilting with purple leaves. Leaves and branches became withered and fibrous roots became brown and rotted. The main roots of severely diseased plants also became rotted. The color of the stem surface turned from red to black, and the color of the stem xylem and phloem turned from dark red to brown. Eventually, the roots of diseased plants became completely rotted and the whole plants became dead, but no stink, which is different from Fusarium solani (Mart.) Sacc. (Yuan et al. 2015). Diseased root tissues (5×5×5 mm in size) were cut from diseased plants, surface-sterilized with 1% sodium hypochlorite for 1 min followed by dipping in 75% alcohol for 30 sec, rinsed in sterile distilled water for 3 times, air-dried on a sterilized filter paper in a laminar flow hood, placed on potato dextrose agar (PDA) containing 250 mg/l of streptomycin sulfate, and incubated at 28℃. Five isolates of Fusarium were obtained and purified using the single-spore isolation method. On PDA plates, the colonies were purple in color with formation of white aerial mycelia and reached 50 to 60 mm in diameter after incubation for 5 days. The colonies produced abundant microconidia on the colonies. The microconidia were 4.3 to 12.3 (10.0) × 2.1 to 3.5 (3.1) µm in size (n = 40), hyaline, ovoid or ellipse in shape. The conidiogenous cells were polyphialides. On mung bean media, the isolates formed macroconidia with 3 to 6 septae, fusiform in shape, slightly curved, 21.8 to 32.7 (31.4) × 2.6 to 4.3 (3.4) µm in size (n = 50). The morphological features of the five isolates were consistent with the description for Fusarium proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg (Leslie and Summerell 2006). To further define the identity of the five isolates, molecular phylogenetic analysis was performed. The genomic DNA was extracted from all five isolates using the cetyl trimethylammonium bromide (CTAB) method. Five genes [nuclear ribosomal internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF1α), ß-tubulin gene, partial sequence for calmodulin (PRO), and RNA-dependent DNA polymerase II subunit (RPB2)] in F. proliferatum were amplified using primers pairs ITS1/ITS4, EF1T/2T, ß-tubulin 2a/b, PRO1/2, and RPB2F/R, respectively (Glass and Donaldson 1995; Liu et al. 1999; Mulè 2004; O'Donnell et al. 1998; O'Donnell et al., 2010). The sequences (GenBank accession numbers: MT371373, MT371384, MT925651, MT925652, and MT934441, respectively) showed 99.6 to 100% identities to the corresponding DNA sequences in F. proliferatum (GenBank Acc. Nos. MK243486, MN245720, KJ12896, MN245721, and MK144327, respectively). All five isolates were tested for pathogenicity to fulfill the Koch's postulates. The 45-day-old healthy plants of S. miltiorrhiza grown in sterilized soil in pots (20 cm in diameter), one plant in one pot, were inoculated with conidial suspensions (1.0 × 107 cfu/ml) by pouring 10 ml conidial suspensions around the stem base in one pot. For each isolate, four plants were inoculated. Four plants were treated with sterilized water in the same volume as a control. The tested plants were placed in a growth room at 25°C (RH > 60%) with a 12 h photoperiod of fluorescent light. The pathogenicity assay was repeated for three times. The similar wilt symptoms were observed on the roots in the inoculated plants 30 days after inoculation but were not observed in the control plants. F. proliferatum was re-isolated from the infected roots, and its identity was confirmed by PCR with the primers described above. To our knowledge, this is the first report of F. proliferatum casing root rot disease on S. miltiorrhiza in China.

Higher plasticity in feeding preference of a generalist than a specialist: experiments with two closely related Helicoverpa species.

Herbivorous insects have been categorized as generalists or specialists depending on the taxonomic relatedness of the plants they use as food or oviposition substrates. The plasticity in host plant selection behavior of species belonging to the two categories received little attention. In the present work, fifth instar caterpillars of the generalist herbivore Helicoverpa armigera and its closely related species, the specialist Helicoverpa assulta, were fed on common host plants or artificial diet, after which their feeding preference was assessed individually by using dual - and triple- plant choice assays. Results show both the two Helicoverpa species have a preference hierarchy for host plants. Compared to the fixed preference hierarchy of the specialist H. assulta, the generalist H. armigera exhibited extensive plasticity in feeding preference depending on the host plant experienced during larval development. Whereas the specialist H. assulta exhibited a rigid preference in both dual and triple-plant choice assays, our findings demonstrate that the generalist H. armigera expressed stronger preferences in the dual-plant choice assay than in the triple-plant choice assay. Our results provide additional evidence supporting the neural constraints hypothesis which predicts that generalist herbivores make less accurate decisions than specialists when selecting plants.

Genetic basis and selection for life-history trait plasticity on alternative host plants for the cereal aphid Sitobion avenae.

Sitobion avenae (F.) can survive on various plants in the Poaceae, which may select for highly plastic genotypes. But phenotypic plasticity was often thought to be non-genetic, and of little evolutionary significance historically, and many problems related to adaptive plasticity, its genetic basis and natural selection for plasticity have not been well documented. To address these questions, clones of S. avenae were collected from three plants, and their phenotypic plasticity under alternative environments was evaluated. Our results demonstrated that nearly all tested life-history traits showed significant plastic changes for certain S. avenae clones with the total developmental time of nymphs and fecundity tending to have relatively higher plasticity for most clones. Overall, the level of plasticity for S. avenae clones' life-history traits was unexpectedly low. The factor 'clone' alone explained 27.7-62.3% of the total variance for trait plasticities. The heritability of plasticity was shown to be significant in nearly all the cases. Many significant genetic correlations were found between trait plasticities with a majority of them being positive. Therefore, it is evident that life-history trait plasticity involved was genetically based. There was a high degree of variation in selection coefficients for life-history trait plasticity of different S. avenae clones. Phenotypic plasticity for barley clones, but not for oat or wheat clones, was frequently found to be under significant selection. The directional selection of alternative environments appeared to act to decrease the plasticity of S. avenae clones in most cases. G-matrix comparisons showed significant differences between S. avenae clones, as well as quite a few negative covariances (i.e., trade-offs) between trait plasticities. Genetic basis and evolutionary significance of life-history trait plasticity were discussed.

Fitness traits and underlying genetic variation related to host plant specialization in the aphid Sitobion avenae.

Sitobion avenae (F.) is an important cereal pest worldwide that can survive on various plants in the Poaceae, but divergent selection on different host plants should promote the evolution of specialized genotypes or host races. In order to evaluate their resource use strategies, clones of S. avenae were collected from oat and barley. Host-transfer experiments for these clones were conducted in the laboratory to compare their fitness traits. Our results demonstrated that barley clones had significantly lower fecundity and tended to have longer developmental times when transferred from barley to oat. However, oat clones developed faster after they were transferred to barley. Clones from oat and barley had diverged to a certain extent in terms of fecundity and developmental time of the nymphs. The separation of barley clones and oat clones of S. avenae was also evident in a principal component analysis. Barley clones tended to have higher broad-sense heritabilities for fitness traits than oat clones, indicating the genetic basis of differentiation between them. Barley clones showed significantly higher extent of specialization compared to oat clones from two measures of specialization (i.e., Xsp and Ysp). Therefore, barley clones were specialized to a certain extent, but oat clones appeared to be generalized. The fitness of S. avenae clones tended to increase with higher extent of specialization. The evolution toward ecological specialization in S. avenae clones, as well as the underlying genetic basis, was discussed.

Differential performance of Sitobion avenae populations from both sides of the Qinling Mountains under common garden conditions.

Sitobion avenae (F.) is a cosmopolitan cereal pest, but geographic barriers like the Qinling Mountains in the Shaanxi Province of China may lead to isolation among its populations, thus causing allopatric speciation. We sampled S. avenae populations from areas north (mean annual temperature, ≍9°C) and south (mean annual temperature, ≍14°C) of the Qinling Mountains, and tested them at 20°C in common garden experiments. The results showed that northern populations had reduced developmental time for first-instar nymphs but prolonged for third- and fourth-instar nymphs compared with southern populations. The postreproductive time and total lifespan of adults from southern populations were longer than those from northern populations, but no significant differences were found in reproductive time or age at first reproduction. Southern populations showed higher lifetime and daily fecundities than northern ones. Significant differences were found in correlation of life-history traits between northern and southern populations. Principal component analyses (PCAs) of S. avenae's vital life-history traits showed separation of populations from three southern locations, indicating their local adaptation. The clustering patterns generated by PCA also showed divergence between northern and southern populations. Alatae of S. avenae seemed to be able to disperse across the Qinling Mountains, which was indicated by the clustering together of some individuals from one side of the mountains with those from the other side. The impacts of the Qinling Mountains on the divergence and gene flow among S. avenae populations, as well as the potential of allopatric speciation for this species, are discussed.

Differential performance of Sitobion avenae (Hemiptera: Aphididae) clones from wheat and barley with implications for its management through alternative cultural practices.

Cultural control measures should play an important role in cereal aphid management on wheat and barley. Planting barley in fields adjacent to those of wheat and intercropping wheat with barley are cultural practices that might suppress the cosmopolitan cereal pest Sitobion avenae (F.). To evaluate the potential effects of these cultural measures on aphid outbreak risks, clones of S. avenae were collected from wheat and barley. Host-switch experiments for the collected clones were conducted in the laboratory to compare their life table parameters and other life history traits. Our results demonstrated that wheat clones of S. avenae had lower developmental times, higher fecundities, and higher growth rates compared with barley clones when tested on the plant of origin. When transferred to barley, the performance of wheat clones was decreased in terms of developmental times of nymphs and age at first reproduction of adults. Barley clones' performance was decreased in terms of reproductive time or lifespan of adult on wheat compared with that on barley. The age-specific survival rates of barley clones tended to be lower than wheat clones in the earlier part of their lives on wheat. The age-specific fecundity of barley clones on barley remained low and fluctuated significantly during their lifetime. Although quite a few significant correlations between developmental and reproductive traits were identified on both plants, no significantly negative correlations (i.e., trade-offs) for the traits were found between plants. The results suggested that barley clones could be generalized but wheat clones were more specialized to their host plant. The potential benefits of the abovementioned cultural measures were discussed.

[Genetic diversity of Rehmannia glutinosa germplasms].

OBJECTIVE: To provide molecular evidences for phylogenetic analysis by studying ITS sequences of Rehmannia glutinosa from different areas. METHOD: The DNAs were extracted from leaves of R. glutinosa by means of CTAB method. The products of PCR amplification were cloned . The data were analyzed by MEGA4.0 software. RESULT: The results showed that the size of the ITS of R. glutionsa tested was from 613 to 614 bp and the length variation was only 1 bp. The sequence of ITS1 was 224-225 bp, and G + C content varied from 60.4% to 63%. The sequence of ITS2 was 224-225 bp and G + C content varied from 57.1% to 65.3%. The sequence of 5. 8S rDNA was 164 bp, it's very conservative in these species. Phylogram tree based on ITS sequence data indicated that the kinship between Bejing No. 2 R. glutinosa and the others were far. There was obvious diversity within wild R. glutinosa varieties, while there was no different among cultivated R. glutinosa varieties. In cultivated R. glutinosa varieties, there was no diversity between R. glutinosa varieties from Henan and those from others provinces. In wild varieties, R. glutinosa from Shengnongshan and Qingtianhe of Henan province showed a closer systematic relationship with cultivated R. glutinosa from Shandong province, while there was no difference between wild R. glutinosa varieties and cultivated varieties from Henan and Shanxi provinces. CONCLUSION: The genetic relationship among R. glutinosa varieties was very close, there was no distinct systematic differentiation.

(E)-2-{4-[1-(Hydroxyimino)ethyl]phenyl-iminomethyl}-6-methoxyphenol mono-hydrate.

In the title compound, C(16)H(16)N(2)O(3)·H(2)O, the benzene rings are nearly coplanar with each other, forming a dihedral angle of 4.46 (3)°. There is a strong intra-molecular O-H⋯N hydrogen bond which results in a six-membered ring. In the crystal, the mol-ecules are connected into a three-dimensional network via O-H⋯O and O-H⋯N inter-molecular hydrogen bonds, forming a centrosymmetric ring along the b axis with graph-set motif R(4) (4)(10). In addition, the short distances between the centroids of six-membered rings [3.555 (1) Å], indicate the existence of π-π stacking inter-actions, which may stabilize the crystal structure.

5,5'-Dimeth-oxy-2,2'-[(nonane-1,9-diyldi-oxy)bis-(nitrilo-methyl-idyne)]diphenol.

The mol-ecule of the title compound, C(25)H(34)N(2)O(6), adopts a fully extended configuration. The oxime (-CH=N-O-) group is coplanar with the aromatic ring and the two benzene rings are almost parallel, making a dihedral angle of 0.16 (3)°. In the crystal structure, strong intra-molecular O-H⋯N hydrogen bonds generate six-membered S(6) ring motifs. Inter-molecular C-H⋯O hydrogen bonds link each mol-ecule to five others, forming an infinite three-dimensional supra-molecular structure. The crystal is further stabilized by π-π stacking inter-actions between neighbouring benzene rings [centroid-centroid distance = 3.744 (2) Å].

1,3-Bis[(4-methylbenzylidene)amino-oxy]propane.

The title bis-oxime compound, C(19)H(22)N(2)O(2), synthesized by the reaction of 4-methyl-2-hydroxy-benzaldehyde with 1,3-bis-(amino-oxy)propane in ethanol, adopts a V-shaped conformation. The dihedral angle between the rings is 84.59 (3)°. The mol-ecule is disposed about a crystallographic twofold rotation axis, with one C atom lying on the axis. In the crystal, mol-ecules are packed by C-H⋯π(Ph) inter-actions, forming chains.