Influence of Chrysanthemum morifolium-maize intercropping pattern on yield, quality, soil condition, and rhizosphere soil microbial communities of C. morifolium.

Introduction: Chrysanthemum morifolium Ramat. is a perennial herb in the Compositae family, often employed in traditional Chinese medicine due to its medicinal value. The planting of C. morifolium faces the challenges of continuous cropping, and intercropping is able to somewhat overcome the obstacles of continuous cropping. Methods: In our study, we designed two different C. morifolium-maize intercropping patterns, including C. morifolium-maize narrow-wide row planting (IS) and C. morifolium-maize middle row planting (IM). Compared with monoculture, the agronomic traits, yield, active ingredients, soil physicochemical properties, soil enzyme activities, and rhizosphere soil microbial communities of C. morifolium and maize were measured under the two C. morifolium-maize intercropping patterns. Results: The findings indicated that (1) Intercropping elevated the agronomic traits, yield, and active ingredients of C. morifolium, especially in C. morifolium-maize narrow-wide row planting pattern, which indicating that interspecific distance played an important role in intercropping system; (2) Intercropping enhanced soil physicochemical properties and enzyme activities of C. morifolium and maize; (3) Intercropping altered rhizosphere soil microbial communities of C. morifolium and maize, making microbial interrelationships more complex. (4) Intercropping could recruit a large number of beneficial microorganisms enrich in the soil, including Bacillus, Sphingomonas, Burkholderia-Caballeronia-Paraburkholderia, Chaetomium, and Ceratorhiza, which may increase the content of AN, NN, AvK, ExCa, AvCu, AvZn and other nutrients in soil and promoted the growth and quality of C. morifolium. Discussion: In summary, intercropping with maize could promote the accumulation of beneficial microorganisms in the soil, thus improving the overall growing environment, and finally realizing the growth and improvement of C. morifolium.

[Identification, biological characterization, and fungicide screening of pathogens causing leaf spot of Belamcanda chinensis].

The leaf spot of Belamcanda chinensis often appears in May to June and spreads rapidly during the flowering stage(July to September) in the cultivation fields, seriously affecting the yield and quality of B. chinensis. To identify and characterize the pathogens of the leaf spot, we isolated two species of Alternaria, identified them according to Koch's postulates, and tested their pathogenicity and biological characteristics. Furthermore, we determined the inhibitory effects of 6 chemical fungicides, 1 plant fungicide, and 3 microbial fungicides on the pathogens by using mycelial growth rate and plate confrontation method to select the appropriate control agents. The results showed that the two pathogens causing B. chinensis leaf spot were Alternaria tenuissima and A. alternata. The conidia of A. tenuissima often formed long chains with no or a few branches, while those of A. alternata often formed short branched chains. The optimum growth temperature of both A. tenuissima and A. alternata was 25 ℃. The two pathogens grew well in alkaline environment. The indoor fungicide screening experiments showed that 40% flusilazole had good inhibitory effects on the two pathogens, with the EC_(50) values of 12.42 mg·L~(-1) and 12.78 mg·L~(-1) for A. tenuissima and A. alternata, respectively. The results of this study provide a theoretical basis for the subsequent theoretical research and field control of B. chinensis leaf spot.

[Identification, biological characteristics, and control of pathogen causing southern blight of Pinellia ternata].

In summer in 2020, Pinellia ternata in many planting areas in Hubei suffered from serious southern blight, as manifested by the yellowing and wilted leaves and rotten tubers. This study aims to identify the pathogen, clarify the biological characteristics of the pathogen, and screen fungicides. To be specific, the pathogen was isolated, purified, and identified, and the pathogenicity was detected according to the Koch's postulates. Moreover, the biological characteristics of the pathogen were analyzed. Furthermore, PDA plates and seedlings were used to determine the most effective fungicides. The results showed that the mycelia of the pathogen were white and villous with silk luster, which produced a large number of white to black brown sclerotia. The pathogen was identified as Athelia rolfsii by morphological observation and molecular identification based on LSU and TEF gene sequences. The optimum growth conditions for A. rolfsii were 30 ℃ and pH 5-8, and the optimum conditions for the germination of sclerotia were 25 ℃ and pH 7-9. Bacillus subtilis, difenoconazole, and flusilazole were identified as effective fungicides with PDA, and their half maximal effective concentration(EC_(50)) was all less than 5 mg·L~(-1). The effective fungicides screened with the seedlings were hymexazol and difenoconazole. Based on the screening experiments, difenoconazole can be used as the main agent for the prevention and treatment of southern blight.

Identifying and characterizing Stagonosporopsis cucurbitacearum causing spot blight on Pinellia ternata in China.

Background: Pinellia ternata (Thunb.), a perennial herbal plant in the Araceae family, has great medicinal value and market demand. In August 2020, an outbreak of severe leaf spot blight disease resulted in a huge yield loss of P. ternata. It is necessary to isolate and identify the pathogens that cause spot blight on P. ternata. Methods: In this study, we isolated and identified the pathogens by fulfilling Koch's postulates. Disease samples with typical spot blight symptoms were collected and pathogens were isolated from the diseased tissues. The pathogen was identified based on its biological characteristics and molecular analysis of internal transcribed (rDNA-ITS) and large subunit (LSU) sequences. Phylogenetic tree were constructed using MEGA7 software and pathogenicity tests were performed using in vivo inoculation. Finally, the pathogen was recovered and identified from the inoculated plants. Results: Based on Koch's postulates, we identified the pathogen causing spot blight on P. ternata as Stagonosporopsis cucurbitacearum. To our knowledge, this is the first study to explore spot blight on P. ternata caused by S. cucurbitacearum in China.

The role of antifungal activity of ethyl acetate extract from Artemisia argyi on Verticillium dahliae.

AIMS: This study investigated the antifungal activity and mechanisms of ethyl acetate extract of Artemisia argyi (EAAA) against Verticillium dahliae. METHODS AND RESULTS: Optical and scanning electron microscopy observation showed that 2.0 mg ml-1 EAAA treatment reduced spore germination rate to 4.56%. Histochemical staining showed that 2.0 mg ml-1 EAAA treatment increased reactive oxygen species (ROS) by more than two times. Physiological test showed that EAAA treatment decreased the contents of soluble proteins and sugars, and reduced the activities of malate dehydrogenase and succinate dehydrogenase by nearly half. Transcriptome analysis showed that EAAA treatment down-regulated the expression of genes involved in primary metabolic pathways of V. dahliae. CONCLUSIONS: Our results revealed that EAAA inhibited the growth and development of V. dahliae from multiple levels and multiple targets, including inhibiting the germination and development of V. dahliae spores, destroying the structure of cell membranes, inducing ROS burst, reducing the activities of respiratory-related enzymes and down-regulating the expression of genes in primary metabolic pathways. SIGNIFICANCE AND IMPACT OF THE STUDY: The mechanism of the multitarget effects of EAAA against V. dahliae may limit the potential of fungus developing resistance and provide the efficient methods to control verticillium wilt disease in the future.

[Identification,biological characteristics and fungicide screening of pathogen of southern blight in Cynanchum stauntonii].

During the high-temperature and rainy season from June to October in 2017-2019,serious southern blight broke out in the Cynanchum stauntonii planting area in Tuanfeng county,Hubei province,which had a great impact on the yield and quality of medicinal materials. In this study,the pathogen of C. stauntonii was isolated,purified,and identified,and the pathogenicity was tested according to Koch's postulates. Meanwhile,the biological characteristics of the pathogen were analyzed. On this basis,the effective fungicides were screened in laboratory. Finally,the pathogen( BQ-1) was identified as Athelia rolfsii( Deuteromycotina,Basidiomycota,anamorph: Sclerotium rolfsii). The optimum growth conditions for BQ-1 were 25-30 ℃,p H 5-8,and alternating light and dark.The effective chemical fungicides were lime-sulphur-synthelic-solution( LSSS) and flusilazole,and the effective botanical fungicide was osthole. BQ-1 was highly homologous to the pathogen HS-1 of peanut southern blight,with the similarity of 18 S r DNA and TEF sequences at 99. 09%. The southern blight in C. stauntonii might be resulted from that in peanut. In the production of C. stauntonii,the following measures should be taken: avoiding rotation or neighboring with peanut,draining water from June to October to reduce humidity,and reasonably applying fungicides.

First Report of Southern Blight on Polygonatum sibiricum Caused by Sclerotium delphinii in China.

Polygonatum sibiricum Delar. ex Redoute is a plant species used for medicine and food. On one hand, its rhizomes have potential medicinal values such as enhancing immunity, anti-aging, anti-tumor and antibacterial as well as the effects of improving memory and reducing blood lipid and sugar. On the other hand, the rhizomes can also be used as raw materials for drinks, preserves, and health products (Su et al. 2018). The annual demand of P. sibiricum is about 3500-4000 tons in China, and the market demands and the price continue to rise in recent years (Su et al. 2018). In August 2019, there was an outbreak of southern blight in the P. sibiricum planting fields (N30°04'06″, E115°39'47″) of Luotian County in Hubei province of China. Approximately 30% of plants were affected in many fields (333.33 ha). We observed that the surface of the infected rhizome and the surrounding soils were covered with white hyphae and sclerotia. The hyphae gradually extended downward to the rhizomes, causing rhizome rot and leaf yellowing and wilting. Mycelial fragments and sclerotia from ten symptomatic rhizomes were collected in the fields and incubated directly on potato dextrose agar (PDA containing 50 µg/ml kanamycin) at 27℃. The fungal colonies were transferred to PDA after two days of cultivation. The white colonies were formed with fluffy aerial mycelia, which grew radially with an average growth rate of 20.54±0.52 mm/d (n=10). The color of the sclerotia was milky white at first, and then gradually turned to beige and yellow-brown. After two-week-incubation, the sclerotia became dark brown. Most of the sclerotia were spherical or nearly spherical, with round-bulges on the surface. The number of mature sclerotia produced per plate ranged from 8-23 (n=10), and the size ranged from 2.5×3.0 mm to 7.5×13.0 mm (5.95 ± 2.34×7.51 ± 2.88 mm; n=50). In addition, clamp connections were observed under the microscope. For molecular identification, genomic DNA was extracted from isolate HJ-1 using the CTAB method (Mahadevakumar et al. 2018). The internal transcribed spacer (ITS) regions of rDNA were amplified with the primers ITS1/ITS4 (White et al. 1990). The resulting showed ITS sequence (Accession number: MW049362) was 99.66% homology with Sclerotium delphinii according to the GenBank database. In addition, the second largest subunit of RNA polymerase II gene (RBP2) and part of the elongation factor 1-alpha (EF1-α) gene were amplified by using the primers RPB26F/RPB2-7CR (Liu et al. 1999) and EF595F/EF1160R, respectively (Wendland and Kothe 1997). RPB2 gene sequence was deposited in GenBank (Accession number: MW415935), and was 99.53% similarity identity to Athelia rolfsii isolate MSB5-1. TEF-1α sequence was deposited in GenBank (Accession number: MW415934), and was 91.35% similarity to S. delphinii strain Sd_405. Because there are very few reference sequences of RPB2 genes from S. delphinii in GenBank to compare, we choose the ITS and TEF-1α gene sequences to construct the concatenated phylogenetic tree by the neighbor-joining method (Tamura et al. 2013). The results showed that HJ-1 was clustered with S. delphinii isolates selected from NCBI database. Based on morphological and molecular characteristics, the fungus was identified as S. delphinii Welch (teleomorph Athelia rolfsii (Curzi) C.C. Tu & Kimbr). Pathogenicity tests were performed on the healthy leaves, roots, stems and plants (n=3) of P. sibiricum. Each sample was inoculated with one sclerotia produced from a fifteen-day-old colony and there was on wound treatment. These inoculated and control samples (treated with sterile water) were incubated in a moist chamber (25 ± 2 °C, RH 85%) (Mahadevakumar et al. 2018). Typical disease symptoms were apparent on leaves, stems, rhizomes and plants at 4, 6, 5 and 15 days post inoculation, respectively. Fulfilling Koch's postulates, the fungal pathogens were isolated and purified from the inoculated site and were reconfirmed as S. delphinii based on the morphological features. To the best of our knowledge, this is the first report of S. delphinii causing southern blight on P. sibiricum in China. S. delphinii has a wide host range worldwide and often causes crop yield reduction. This study will be helpful for the prevention and control of P. sibiricum southern blight in the future.

[Identification and biological characteristics of southern blight causing root rot on three medicine plants of Iridaceae in Dabie Mountains].

In order to identify the species and biological characteristics of the pathogen of southern blight from three kinds of Chinese medicine of Iridaceae(Belamcanda chinensis, Iris tectorum and I. japonica) in Dabie Mountains, the isolation, identification, pathogenicity and biological characteristics of the pathogens were studied according to Koch's postulates. In addition, 9 chemical fungicides, 3 botanical fungicides and 5 microbial fungicides were used to evaluate their inhibition to the isolates in vitro. The results showed that all the strains(SG-Q, YW-Q, and HDH-Q) isolated and purified from the diseased plants of B. chinensis, I. tectorum and I. japonica, respectively, were identified as Sclerotium rolfsii through morphological observation and sequence aligement of 18 S rDNA, rDNA-ITS and TEF. Field observations showed that the intensity of the disease incidence of three Iridaceae plants was B. chinensis>I. japonica> I. tectorum, and the pathogenicity of the strains was SG-Q>YW-Q>HDH-Q. For biological characteristics, SG-Q strain was suitable for growth under the 12 h light/12 h dark cycle, with the optimal growth temperature of 30 ℃ and pH of 5. Among the 9 tested chemical fungicides, 29% lime sulphure and 10% flusilazole had stronger inhibitory effect on mycelia growth of SG-Q. For 3 botanical fungicides, 1% osthol, 20% eugenol and 0.5% berberine could effectively inhibt the mycelial growth of SG-Q and cause the morphological variation of the pathogen. For 5 microbial fungicides, Trichoderma harzianum and Bacillus subtilis had better inhibition on the mycelium growth of SG-Q.

Allelopathic effect of Artemisia argyi on the germination and growth of various weeds.

Allelopathy means that one plant produces chemical substances to affect the growth and development of other plants. Usually, allelochemicals can stimulate or inhibit the germination and growth of plants, which have been considered as potential strategy for drug development of environmentally friendly biological herbicides. Obviously, the discovery of plant materials with extensive sources, low cost and markedly allelopathic effect will have far-reaching ecological impacts as the biological herbicide. At present, a large number of researches have already reported that certain plant-derived allelochemicals can inhibit weed growth. In this study, the allelopathic effect of Artemisia argyi was investigated via a series of laboratory experiments and field trial. Firstly, water-soluble extracts exhibited the strongest allelopathic inhibitory effects on various plants under incubator conditions, after the different extracts authenticated by UPLC-Q-TOF-MS. Then, the allelopathic effect of the A. argyi was systematacially evaluated on the seed germination and growth of Brassica pekinensis, Lactuca sativa, Oryza sativa, Portulaca oleracea, Oxalis corniculata and Setaria viridis in pot experiments, it suggested that the A. argyi could inhibit both dicotyledons and monocotyledons not only by seed germination but also by seedling growth. Furthermore, field trial showed that the A. argyi significantly inhibited the growth of weeds in Chrysanthemum morifolium field with no adverse effect on the growth of C. morifolium. At last, RNA-Seq analysis and key gene detection analysis indicated that A.argyi inhibited the germination and growth of weed via multi-targets and multi-paths while the inhibiting of chlorophyll synthesis of target plants was one of the key mechanisms. In summary, the A. argyi was confirmed as a potential raw material for the development of preventive herbicides against various weeds in this research. Importantly, this discovery maybe provide scientific evidence for the research and development of environmentally friendly herbicides in the future.

First Report of Fusarium wilt of Coleus forskohlii Caused by Fusarium oxysporum in China.

Coleus forskohlii (Wild) Briq. is an aromatic plant in the Lamiaceae family cultivated primarily in India, Sri Lanka, Nepal and China (Yunnan Province). This herb is considered to have medicinal properties and the whole plant can be used to treat asthma, cancer and other diseases with remarkable efficacy. Due to the high medicinal and economic value of C. forskohlii, it has been introduced to Tongcheng (N29°18'12.24″, E113°53'59.36″), Hubei Province for cultivation. However, severe Fusarium wilt disease of C. forskohlii has been epidemic in Tongcheng since 2018 with a disease incidence of 5 to 30% in surveyed fields. This disease is characterized typically by root rot, vascular discoloration and leaf wilting of C. forskohlii (Fig 1), resulting in progressive plant death. Ten diseased plants were collected from the fields and the roots and stems were rinsed in 70% ethanol for 5 min and samples at the junction of disease and healthy tissues (0.5 × 0.5 cm2) were cutted and placed on potato dextrose agar (PDA) for fungal isolation in a dark chamber at 28°C. Eventually, ten pure isolates were obtained from hyphal-tip followed by single-spore purification on PDA. Seven of the purified isolates showed white aerial mycelium initially and secreted orange-brown pigment 8 days after incubation. Macroconidia were falciform, hyaline, three to five septate, ranging from 2.02 to 4.17 (mean 2.98 µm) × 10.05 to 21.90 µm (mean 12.04 µm) in size (n = 30) (Fig 2). These morphological characteristics resembled Fusarium oxysporum. (Leslie and Summerell 2006) and we selected one of them for molecular identification. Genome DNA was extracted from isolate (RS-4) using the CTAB method (Mahadevakumar et al. 2018). The translation elongation factor 1 alpha (EF-1α) DNA sequence was amplified using primers EF1/EF2 (Geiser et al. 2004), and the second largest subunit of RNA polymerase II (RPB2) DNA sequence was amplified using primers fRPB2-5F/fRPB2-7cR (Liu et al. 1999). The obtained EF-1α sequence of RS-4 (MW219142) showed 100% identity with that of F. oxysporum (FD_01376) (FUSARIUM-ID database). RPB2 sequences of RS-4 (MW219143) showed 100% identity with F. oxysporum (FD_01679) (FUSARIUM-ID database). Moreover, a phylogenetic tree of the EF-1α gene sequence of RS-4 was constructed based on the Neighbor-Joining method in MEGA7 software (Tamura et al. 2013) and revealed that strain RS-4 was closest to F. oxysporum (Fig 2). To test the pathogenicity of RS-4, six healthy leaves of C. forskohlii were collected and inoculated either with the colonized PDA discs (diameter, 5 mm) of RS-4 or control PDA discs, in a moist chamber at 25 ± 2°C. Five days later, brown-black lesions were observed on all inoculated leaves. However, the non-inoculated leaves were maintained asymptomatic. For in vivo pathogenicity test, twenty-day-old C. forskohlii plants (n=3) were inoculated with 106 spores/ml of the RS-4 at a position approximately 1 cm above the soil. Three seedlings treated with sterile water were used as controls. These inoculated and control seedlings were incubated in a moist chamber (25 ± 2 °C, RH 85%). Three days later, typical Fusarium rot symptoms were observed on all inoculated seedlings with rotten stems and withering leaves (Fig 2). Fungal pathogens were re-isolated from the inoculated sites of in vitro and in vivo inoculations by repeating the above isolating operation, and were reconfirmed through morphological features. This is the first report of F. oxysporum causing root rot on C. forskohlii in China. F. oxysporum is one of the most economically important fungal pathogens causing vascular wilt on a wide range of plants worldwide (Dean et al. 2012). The identification of F. oxysporum as the causal agent of the observed Fusarium wilt on C. forskohlii, is critical to the prevention and control of this disease in the future. Acknowledgement This research was supported by funding from the Key project at the central government level titled, "The ability to establish sustainable uses for valuable Chinese medicinale resources" (2060302) Reference Dean, R., et al. 2012. Mol. Plant. Pathol. 13: 414. https://doi.org/10.1111/j.1364-3703.2011.00783.x. Geiser, D. M., et al. 2004. Eur. J. Plant Pathol. 110: 473. https://doi.org/10.1023/B:EJPP.0000032386.75915.a0. Leslie, J. F. and Summerell, B. A. 2006. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, U.K. Liu, Y. J., et al. 1999. Mol. Biol. Evol. 16: 1799. https://doi.org/10.1093/oxfordjournals.molbev.a026092 Mahadevakumar, S. et al. 2018. Eur. J. Plant Pathol. 151:1081. https://doi.org/10.1007/s10658-017-1415-2. Tamura, K., et al. 2013. Mol. Biol. Evol. 30: 2725. https://doi.org/10.1093/molbev/msw054.

First Report of Leaf Spot on White Chrysanthemum (Chrysanthemum morifolium) Caused by Epicoccum sorghinum in Hubei province, China.

White Chrysanthemum (Chrysanthemum morifolium), a perennial herb of the Compositae family, is used for traditional medicine. The planting area of white chrysanthemum in Macheng city, Hubei Province is about 3333 ha and the annual output can reach more than 5000 tons. In 2019, leaf spot disease appeared on almost all middle and lower leaves of white chrysanthemum in most fields of Fengshumiao county, Macheng city (N31°29'57″, E115°05'49″). This county has 33 acres white chrysanthemum planting area, and most of the plants in the county were infected with the leaf spot disease. The average incidence of leaf spot disease was 65%, and incidence in some areas was 100%. In our observations, leaf spot disease can occur throughout the whole growth period of white chrysanthemum, and it will become more serious under the high temperature and humidity condition. Usually, the diseased leaves account for 30 to 80% of the total leaves on the plant. Leaf spot initially manifests as necrotic lesions on the edge and tip of the leaf, and then the lesions coalesce and gradually expand to form irregular light-brown to brown-black spots, eventually leading to necrosis and curling of the entire leaf. This disease seriously affects the growth and development of plants, resulting in the decline of yield and quality of white chrysanthemum. Ten symptomatic leaf samples were collected, the surfaces were disinfected with 0.1% mercuric chloride (HgCl2) for 3 min, and washed with sterile distilled water three times. Ten tissue samples at the junction of diseased and healthy areas (0.5 × 0.5 cm2) were cut and placed on potato dextrose agar (PDA) medium containing 100 µg/ml cefotaxime sodium and incubated in a dark chamber at 28°C. After 2 days, the hyphal tips from the edges of growing colonies were transferred to fresh PDA plates for further purification. Finally, eight isolates were obtained and these isolates were similar in morphology. The color of purified isolates was initially white to pale yellow. After six days of incubation, colonies had a diameter of 8 cm and the cultures were pale gray and starting to secrete scarlet pigment. After 15 days incubation, the colonies were grayish brown, while the backside was reddish-brown. Gray to tan chlamydospores were observed, nearly spherical, with a wart-like surface. Unicellular chlamydospores were 7.91 to 32.23 × 12.03 to 38.42 µm (n=30) and multicellular chlamydospores were 6.32 to 25.10 × 21.75 to 100.05 µm (n=30). The morphological characteristics were similar to Epicoccum sorghinum (Kang et al. 2019). The isolate FDY-5 was chosen for molecular identification. The sequence of rDNA-ITS, TUB, and LSU of the FDY-5 were amplified (GenBank MT800929, MT799852, and MT800935, respectively) (White et al. 1990; Carbone and Kohn 1999; Lumbsch et al. 2000). BLAST results showed that the rDNA-ITS sequences, the TUB gene sequences, and LSU gene sequences of strain FDY-5 shared 99% identity with the sequences of E. sorghinum (syn. Phoma sorghina) in GenBank (MN555348.1, MF987525.1, MK516207.1, respectively). Moreover, a phylogenetic tree of the LSU gene sequence of FDY-5 was constructed based on the Neighbor-Joining (NJ) method in MEGA6 software (Tamura et al. 2013) and revealed that strain FDY-5 was closest to E. sorghinum. Based on morphological and molecular characteristics, the fungus was identified as E. sorghinum. Pathogenicity tests were conducted on two-month-old white chrysanthemum plants. The upper three leaves of three plants were randomly selected for stab treatment and were inoculated with 5 × 5 mm mycelial discs produced from a fifteen-day-old colony on PDA. The inoculated and control (treated with sterile PDA disks) plants were incubated in a moist chamber (25 ± 2 °C, RH 85%). The first lesions appeared 1 day after inoculation on leaves, and the necrotic lesion area expanded outward and showed typical symptoms 3 days later. To fulfill Koch's postulates, the pathogen was reisolated from nine inoculated leaves by repeating the above isolating operation, and confirmed as E. sorghinum by morphology. To the best of our knowledge, this is the first report of E. sorghinum causing leaf spot on white chrysanthemum in China. E. sorghinum has a wide host range worldwide and often causes crop yield reduction. This report will facilitate the diagnosis of white chrysanthemum leaf spot of white chrysanthemum allowing control measures to be adopted to manage this disease in a timely manner. References Carbone, I., and Kohn, L. M. 1999. Mycologia 91:553. Kang, Y., et al. 2019. Plant Dis. 103 (7):1787. Lumbsch, H., et al. 2000. Plant Biol. 2:525. Tamura, K., et al. 2013. Mol. Biol. Evol. 30:2725-2729. White, T. J., et al. 1990. Page 315 in:PCR protocols:a guide to methods and applications. Academic Press, San Diego, CA. Funding Funding was supported by Major Increase and Decrease Projects at the Central Level of China (2060302) and the National Key Research and Development Program (2017FYC1700704).