The AA9 LMPO protein, CcAA9-20333, acts as a secretory effector by inhibiting N. benthamiana cell death via its enzymatic activity and contributes to the virulence of C. coccodes. Although the functions of most effectors produced by fungi are unclear, it is known that they may both increase infection and alter host immunity. This research describes a novel fungal effector protein CcAA9-20333 of Colletotrichum coccodes. First, the signal peptide of CcAA9-20333 has secretory function verified by yeast trap secretion assay and subcellular localization, and CcAA9-20333 with the full length can inhibit host immunity including the PCD induced by INF1 and BAX, accumulation of ROS, ion leakage, and genes’ expression involved in SA and JA pathway, and even inhibit the susceptibility of plant to C. coccodes. The expression level of CcAA9-20333 was associated with the regulation mechanism of it on immune response, with the positive correlation. In addition, all of these functions were decided by the enzymatic activity of CcAA9-20333, which was verified by site mutant. The morphological characteristics of ΔCcAA9-20333, the mutant of C. coccodes, demonstrated that CcAA9-20333 can reduce the sporulation and virulence. All of these provided that CcAA9-20333 play a critical role in regulating the immunity of host plant and pathogenicity of C. coccodes. This approach teaches us to see beyond the obvious while expanding the field of LPMO study.

Auxiliary activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade a variety of polysaccharides. Despite extensive research on this class of enzymes, the essential LPMO gene involved in pathogenicity and its characterization still requires further study. Here, we obtained the candidate effector AA9 LPMOs gene CcAA9-20333 by mining transcriptome data of Colletotrichum coccodes, the causal agent of potato anthracnose. A novel CcAA9-20333 gene was cloned, prokaryotically expressed, purified, and characterized. Results indicated that the CcAA9-20333 exhibits optimal activity at 40-60 °C and has high thermostability remaining active even exposed to 60 °C for 80 min. The highest CcAA9-20333 enzymatic activity was detected at pH 6.0 and excellent pH stability was observed after incubation at pH 6.0 for 24 h at 4 °C. CcAA9-20333 crude enzyme released reducing sugars in the presence of several different metal ions and exhibited substrate specificity. Four mutants (M1-M4) were obtained with one-step point mutation methodology using pET30a(+)-CcAA9-20333 as a template. A pathogenicity assay indicated that the potential pathogenicity of wild-type to Solanaceae plants was stronger than any of the mutant strains. The gene CcAA9-20333 was cloned, heterologously expressed, and potential substrate binding sites were characterized using site-directed mutation methodology. This is the first report on the LPMO gene CcAA9-20333 from C. coccodes and its role in pathogenicity.

Recently, a new bacterial disease was detected on cucumber stalks. In order to study the pathogenesis of this disease, the pathogenic bacteria were isolated and identified on the basis of morphological and molecular characteristics, and further analyzed for pathogenicity and antagonistic evaluation. Pathogenicity analysis showed that HlJ-3 caused melting decay and cracking in cucumber stems, and the strain reisolated from re-infected cucumber stalks was morphologically identical to HlJ-3 colonies, which is consistent with the Koch’s postulates. The pathogenic strain HlJ-3 was identified as having similar morphological characteristics to Bacillus subtilis. Meanwhile, its internal transcribed spacer sequence (ITS) and DNA gyrase A subunit (gyrA) were both more than 99% homologous and clustered on the same branch with B. subtilis. Therefore, combined with morphological and molecular biological features, strain HlJ-3 was identified as B. subtilis. In addition, B. subtilis, which has a wide range of hosts, was able to infest other common crop species, including potato, tomato, pepper, melon, and radish. Furthermore, antagonistic evaluation confirmed that strain HlJ-3 strongly inhibited the mycelial growth of Colletotrichum coccodes and Alternaria tenuissima in vitro, with antagonistic effects of 69.92% and 68.08%, respectively. In conclusion, our results showed that strain HlJ-3 is B. subtilis, which is pathogenic to cucumber in vivo and can infect plants of Solanaceae, Cucurbitaceae and Brassicaceae with a wide range of hosts. In addition, this strain has good biocontrol effects against C. coccodes and A. tenuissima in vitro. The findings of this research will help to prevent and control the occurrence of this pathogen and regulate its use as a biocontrol agent.

Pectate lyases (PL), as important polysaccharide lyases, play an important role in the infection of host plants by pathogenic. A previous study found that the PL gene CP966_RS08110 was up-regulated in the interaction between Streptomyces galilaeus 5T-1 and potatoes. In this study, S. galilaeus 5T-1 was used as the study object, and its gene function was investigated using bioinformatics analysis, prokaryotic expression, and CRISPR-Cas9 technology. The previous results showed that the pectate lyase CP966_RS08110 gene of Streptomyces galilaeus 5T-1 was up-regulated in the pathogenic process. In this study, the CP966_RS08110 gene was cloned from the genomic DNA of S. galilaeus 5T-1. It encoded for a 415-residue protein with a complete PL-6 superfamily domain and Pec_lyase_C domain, which belongs to the PL1 family. The soluble protein encoded by CP966_RS08110 was obtained successfully, which has high pathogenicity after inoculating healthy potatoes. The mutant strain △PL5T-1 with CP966_RS08110 gene deletion was successfully obtained, and its colony morphology and pigment were not significantly different from that of wild strains, but its growth rate was slowed down, moreover, the hyaline circle formed by the mutant strain ΔPL5T-1 using pectin was significantly smaller than wild strain, and the deletion of this gene affected the infestation rate of S. galilaeus 5T-1. Our results confirm that the CP966_RS08110 gene was the pathogenic factors and played a key role in process of infecting and causing potato common scab, which laid foundation for understanding the pathogenic mechanism of S. galilaeus 5T-1.

In June 2020, branch canker and brown spot of leaves on walnut were observed in Qingyang, China. A total of five strains were isolated from symptomatic samples and verified for pathogenicity based on Koch's Postulates, which showed that strains Hz4-1, Hz5-2, and Hy3-1 were the causative agents, and preliminarily identified them as members of the genus Nothophoma based on morphological characteristics. Subsequently, multi-locus phylogenetic analysis of the partial sequences of internal transcribed spacer (ITS), nuclear large ribosomal subunit (LSU), partial beta-tubulin (TUB2), and RNA polymerase II second largest subunit (RPB2) showed that the three strains were clustered into two groups on the phylogenetic tree: strain Hz4-1 and Hz5-2 and Nothophoma quercina (CBS 6633.92) clustered into one branch with 84% support, while Hy3-1 and Nothophoma spiraeae (CFCC 53 929) clustered within a well-supported clade with a bootstrap value of 99%. As a result, combining morphological characteristics and multi-locus systematic analysis, the strains Hz4-1 and Hz5-2 were identified as N. quercina, and Hy3-1 was identified as N. spiraeae, which is the first report of N. spiraeae causing walnut disease in China.

Brown spots occurring on potato leaves affect the normal growth of host plants. Recently, a new brown spot disease with some black dots was discovered on potato leaves in Dingxi, China. To confirm the causal agents of potato brown spots, the diseased lesions were collected and disinfected, and only one type of isolate, with football-shaped conidia, was obtained from disinfected diseased leaves in three experimental fields. Pathogenicity tests of four representative isolates revealed that they can cause disease symptoms on 50%-75% of wounded and non-wounded potato leaves. In addition, a typical isolate, DXPH, was identified based on morphological and molecular characteristics, and the colony and conidial characteristics were similar to those of Epicoccum sp. Multilocus sequence analysis showed that the internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2) and beta-tubulin (Tub2) genes of DXPH were 100%, 100% and 99% identical to the sequences of Epicoccum nigrum (CBS 140523), respectively. Additionally, antagonistic evaluation confirmed that the representative pathogenic strain DXPH showed greatest inhibition of the mycelial growth of Colletotrichum coccodes and Fusarium chlamydosporum in vitro, with antagonistic effects of 66.67% and 61.56%, respectively. Our findings provide evidence that E. nigrum can be pathogenic to potatoes in vivo in China, while retaining a biocontrol capacity on C. coccodes, Fusarium spp. and Botrytis cinerea in vitro, which will be helpful for the effective control of potato brown spot disease and management of the use of E. nigrum as a biological control agent.

Volatile organic compounds (VOCs) produced by endophytic strains play important roles in enhancing plant resistance to pathogens; however, the VOCs produced Bacillus mojavensis ZA1 on the inhibiting ability of Colletotrichum coccodes remain unknown. Hence, the effects of VOCs in controlling potato anthracnose (C. coccodes) were investigated. Alcohols, ketones, phenols, and esters were antifungal volatile compounds in the volatile organic compounds (VOCs) produced by B. mojavensis ZA1 after fermentation. The number of VOCs was 29 and 49 at extraction temperatures of 28 °C and 40 °C, respectively. The antifungal activity of 13 of these compounds was tested, and the results showed that 2-ethyl-1-hexanol completely inhibited mycelial growth, sporulation, and conidial germination of C. coccodes. The antifungal activity of VOCs; 2-methyl-1-butanol, 4,4′-(1-methylethylidene) bis-phenol, 2-nitro-ethanol, 2,3-butanediol and 3-methyl-1-butanol was also impressive; not only did they inhibit mycelial growth and spore germination by more than 43%, but they also reduced the pathogen’s conidia production by 43-fold. After studying the structure of organic volatiles with high fungistatic activity, it was predicted that the hydroxyl group could be an effective functional group for B. mojavensis ZA1 to achieve fungal suppression. The antifungal activity of metabolites of alcohols and ketones with 4–8 carbon atoms was superior against pathogenic fungus. As a result, carbon-chain length has been proposed as one of the parameters regulating the fungistatic efficacy of secondary metabolites. By utilizing metabolites produced by B. mojavensis ZA1, this research lays the groundwork for using B. mojavensis ZA1 in biological control.

Background Pepper is a popular ingredient in many Chinese households; however, anthracnose caused by Colletotrichum spp. has greatly decreased pepper production. The genus Bacillus is widely known for its important role in the development and protection of plants from phytopathogenic fungi. Results Fifty-eight endophytic strains were isolated from pepper leaves and tested for antifungal activity in this study. Specifically, L1-7 and L3-5 displayed growth inhibition rates of 79 and 80% against C . scovillei mycelium, respectively, while 25 of these strains all had growth inhibition rates of greater than 60%. Bacillus amyloliquefaciens and B. velezensis , based on culture and morphological identification with 16S rDNA and gyrB gene sequence analyses, were determined to be the respective species L1-7 and L3-5. Additionally, it was discovered that these two antagonistic endophytic bacteria could fix nitrogen, produce indoleacetic acid (IAA) and have a high salt tolerance. Pot experiments again showed excellent control of the pathogen C . scovillei by L1-7 and L3-5, with 80.64 and 73.39% control, respectively. Therefore, B . amyloliquefaciens (L1-7) and B . velezensis (L3-5) can be applied as biological control agents to protect peppers against C . scovillei -caused anthracnose. Conclusion Bacillus amyloliquefaciens (L1-7) and B . velezensis (L3-5) can be applied as biological control agents to protect peppers against C . scovillei -caused anthracnose. Thus, they can serve as promising biocontrol agents and plant growth promoters, and future research on the pertinent bacteria will serve as a useful guide for the creation of microbial resources.

以尖镰孢菌黄瓜专化型(Fusarium oxysporum f.sp.cucumerium)为指示菌从青海高寒草地牧草内生细菌中筛选拮抗菌株,结合形态学特征和16Sr DNA基因序列同源性分析鉴定,并进行促生功能测定.结果表明,供试的81株内生细菌中36株有拮抗作用,占供试菌株的44.0％,其中从根部分离的13株的抑菌率均在50.0％以上,菌株2018GH11对6属8种病原真菌的抑菌率均在50.0％以上,特别对孜然根腐病菌的抑菌率达67.6％,抑菌谱宽;根据形态学特征和16SrDNA基因序列分析将其鉴定为暹罗芽孢杆菌Bacillus siamese;2018GH11具有固氮能力,无溶磷和产IAA能力;发酵菌剂2018GH11用量为0.50％时,黄瓜株高、茎粗、叶片数、鲜重及干重分别比对照增加了 15.33％、39.40％、22.22％、215.99％和126.70％.该结果表明高寒草地牧草内生细菌2018GH11具有良好的防病促生作用,开发潜力巨大.

在甘肃省天水市林区采集到一种症状与已报道略有差别的云杉叶枯病,对病原菌进行分离与鉴定,并测定其致病性和生物学特性.结果表明,在云杉上引起叶枯病症状的分离物SJ1菌丝呈白色、红橙色、深黄棕色,后期菌落表面出现红黑色液滴,分生孢子黑色,扁圆形或近球形,表面有褶皱.ITS、TUB2和RPB2基因序列分析表明其与黑附球菌(Epicoccum nigrum)同源性最高,且在系统发育树上聚在一起,因此将其鉴定为黑附球菌(E.nigrum).生物学特性测定结果表明,最适菌丝生长的培养基为麦芽浸粉琼脂培养基,温度为25℃,pH为7,光照对菌丝生长影响不大,最适碳、氮源分别为淀粉和蛋白胨,氮源为尿素时不生长.

The plant disease Colletotrichum coccodes, which lowers potato yields, poses a severe danger to the booming potato industry. Isolated plant endophytic bacteria from highland pasture can produce a variety of metabolites that lessen the risk that the pathogen C. coccodes poses to plant growth and development. Therefore, the objective of our work was to assess substances with antipathogenic properties made by the endophytic bacteria Bacillus mojavensis ZA1. Gas chromatography-mass spectrometry (GC-MS) was used in our investigation to accomplish a thorough structural elucidation of the antipathogenic compounds produced by the endophytic bacterial strain B. mojavensis ZA1. The results showed that the metabolites extracted from ethyl acetate as an extractant were the most effective in inhibiting the pathogen C. coccodes, with 60.95% inhibition. Thirty-five distinct chemicals, including acids, esters, ketones, alcohols, amino acid ammonium salts, cyclic ethers, aromatic hydrocarbons, and heterocyclic compounds, were among the metabolites that may inhibit C. coccodes. Further analysis of the chemical groups in the compound structures revealed the potential of driving groups, such as hydroxyl, carbonyl, ester, benzene, carbon-carbon double bonds, and carbon rings, that prevent C. coccodes from performing its function. This study opens up new opportunities for plant protection programs by demonstrating that natural chemicals produced by B. mojavensis ZA1 can be used as candidates for cutting-edge plant disease management treatments.

Picea asperata is a unique tree species in China. It has important economic and ecological values. In this study, the disease symptoms resembling needle blight, a new disease of P. asperata (10- to 15-year-old), were detected in a forest area in Tianshui City, Gansu Province, China, in August 2020. Affected needles turned yellow, the colour became darker at the junction of infected and healthy areas, the needles wilted and even fell off the tree. Through pathogenicity tests, the two pure fungi strains isolated from diseased needles caused distinct needle blight symptoms on P. asperata, with symptoms similar to those observed in the forest. It was observed that they had sickle-shaped conidia. Subsequently, multilocus phylogenetic analysis of partial sequences of the internal transcribed spacer region (ITS), translation elongation factor 1-alpha (TEF-1 alpha) and RNA polymerase II second major subunit (RPB2) genes was performed. They were found closely clustered with Fusarium oxysporum and F. avenaceum, respectively. Based on morphological and molecular biological determinations, the two pathogens were identified as F. oxysporum and F. avenaceum. To our knowledge, this is the first report of needle blight caused by F. oxysporum and F. avenaceum on P. asperata in China.

Despite paying little attention to the functional groups of its metabolites for anti-pathogen, Bacillus mojavensis ZA1 has been an excellent functional strain for suppressing pathogens. This study sought to understand the major functional groups that contribute to inhibiting Colletotrichum coccodes in the metabolites as well as the lipid metabolome of B. mojavensis ZA1. In our research, sixty anti-pathogen fractions were obtained by separation using chromatographic columns, in which the fraction with the best effect reached 74% pathogen inhibition rate. Thin layer chromatography was used to separate the inhibitory fractions into two groups, BZS1 and BZS2, for a total of 8 fractions. The non-targeted metabolome was detected by ultraperformance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. A total of 247 lipid species, comprising 135 species in the positive mode and 112 species in the negative mode, were found using non-targeted metabolomics. They were primarily enriched in 12 metabolic pathways, 10 of which had ester groups in the structural formulae of all enriched compounds, while the other two, sphingomyelin metabolism (positive mode) and sterol metabolism (negative mode), had carbonyl and steroid nucleus in the structural formulae of all enriched compounds, respectively. In conclusion, the predominant anti-pathogen functional groups in the lipid metabolites produced by B. mojavensis ZA1 are ester groups, carbonyl groups, and steroid nucleus. This information will build a solid foundation for the employment of B. mojavensis ZA1 as biocontrol agents for C. coccodes and will provide more thorough and favorable data in support of that claim.

Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum is a serious fungal, soil-borne disease that has a dramtic negative impact on the yield and quality of cucumber. In this study, isolates of endophytic bacteria isolated from healthy cucumber plants were evaluated for their ability to inhibit Fusarium wilt. Results indicated that 6 out of 100 of the obtained endophytic bacterial isolates exhibited significant inhibitory activity against mycelial development of F. oxysporum f. sp. cucumerinum, with an inhibition rate > 60 % in dual culture assays. Among the active isolates, strain hg18 exhibited the strongest activity with a 69.57 % rate of inhibition. Strain hg18 was identified as Paenibacillus polymyxa based on morphological, physiological, and biochemical traits, as well as a phylogenetic tree that was constructed using 16S rRNA and rpoB gene sequences. Biological and PCR assays demonstrated that strain hg18 can secrete IAA, a siderophore, hydrolytic enzymes, including protease, cellulase and glucanase, and antimicrobial compounds, such as iturin, fengycin, and non-ribosomal polypeptide synthase, as well as fix nitrogen. Strain hg18 decreased the disease index of cucumber Fusarium wilt and promoted cucumber growth in non-diseased plants in greenhouse experiments, compared to a water treatment. These findings demonstrate the potential of using P. polymyxa hg18 as a biocontrol agent for controlling Fusarium wilt in cucumber.

Biotic stress deleteriously affects growth, development, and productivity in plants. Proline (Pro) plays a signif-icant role in enhancing plant resistance to pathogen infection. However, its effects on reducing Lelliottia amni-gena-induced oxidative stress in potato tubers remain unknown. The present study aims to evaluate the in vitro Pro treatment in potato tubers exposed to a newly emerging bacterium, L. amnigena. Sterilized healthy potato tubers were inoculated with 0.3 mL of L. amnigena suspension (3.69 x 107 CFU mL-1) 24 h before Pro (5.0 mM) application. The L. amnigena treatment significantly increased the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in the potato tubers by 80.6 and 85.6%, respectively, compared to the control. Application of proline (Pro) decreased MDA and H2O2 contents by 53.6 and 55.9%, respectively, compared to the control. Application of Pro to L. amnigena-stressed potato tubers increased the activities of NADPH oxidase (NOX), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO), phenylala-nine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), 4-coumaryl-CoA ligase (4CL) and cinnamate-4-hydroxylase (C4H) C4H by 94.2, 96.3, 97.3, 97.1, 96.6, 79.3, 96.4, 93.6, and 96.2%, respectively, compared to the control. In comparison to the control, the genes PAL, SOD, CAT, POD, and NOX were signifi-cantly increased in the Pro-treated tubers at 5.0 mM concentration. Tubers treated with Pro + L. amnigena increased the transcript levels of PAL, SOD, CAT, POD, and NOX by 2.3, 2.2, 2.3, 2.5, and 2.8-fold respectively, compared to the control. Our findings suggested that pretreatment of tubers with Pro might reduce lipid per -oxidation and oxidative stress by enhancing enzymatic antioxidant activity and gene expression.

HomePlant DiseaseVol. 106, No. 5First Report of Botrytis cinerea Causing Gray Mold on Astragalus membranaceus in China PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Botrytis cinerea Causing Gray Mold on Astragalus membranaceus in ChinaMengjun Jin, Chengde Yang, Lijuan Wei, Lingxiao Cui, Yidan Wang, and Richard OseiMengjun Jinhttps://orcid.org/0000-0001-6485-1734College of Plant Protection, Gansu Agricultural University, Lanzhou 730070, ChinaBiocontrol Engineering Laboratory of Crop Diseases and Pests of Gansu Province, Lanzhou 730070, ChinaSearch for more papers by this author, Chengde Yang†Corresponding author: C. D. Yang; E-mail Address: yangcd@gsau.edu.cnhttps://orcid.org/0000-0003-4942-8959College of Plant Protection, Gansu Agricultural University, Lanzhou 730070, ChinaBiocontrol Engineering Laboratory of Crop Diseases and Pests of Gansu Province, Lanzhou 730070, ChinaSearch for more papers by this author, Lijuan WeiCollege of Plant Protection, Gansu Agricultural University, Lanzhou 730070, ChinaBiocontrol Engineering Laboratory of Crop Diseases and Pests of Gansu Province, Lanzhou 730070, ChinaSearch for more papers by this author, Lingxiao Cuihttps://orcid.org/0000-0002-8460-6493College of Plant Protection, Gansu Agricultural University, Lanzhou 730070, ChinaBiocontrol Engineering Laboratory of Crop Diseases and Pests of Gansu Province, Lanzhou 730070, ChinaSearch for more papers by this author, Yidan WangCollege of Plant Protection, Gansu Agricultural University, Lanzhou 730070, ChinaBiocontrol Engineering Laboratory of Crop Diseases and Pests of Gansu Province, Lanzhou 730070, ChinaSearch for more papers by this author, and Richard Oseihttps://orcid.org/0000-0003-4634-8339College of Plant Protection, Gansu Agricultural University, Lanzhou 730070, ChinaBiocontrol Engineering Laboratory of Crop Diseases and Pests of Gansu Province, Lanzhou 730070, ChinaSearch for more papers by this author AffiliationsAuthors and Affiliations Mengjun Jin1 2 Chengde Yang1 2 † Lijuan Wei1 2 Lingxiao Cui1 2 Yidan Wang1 2 Richard Osei1 2 1College of Plant Protection, Gansu Agricultural University, Lanzhou 730070, China 2Biocontrol Engineering Laboratory of Crop Diseases and Pests of Gansu Province, Lanzhou 730070, China Published Online:30 Mar 2022https://doi.org/10.1094/PDIS-06-21-1330-PDNAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleAstragalus membranaceus, known as Huang-qi, is a perennial herbal medicine plant that grows 50 to 150 cm high, with 13 to 31 small leaves (Fu et al. 2014). In September 2019, a disease investigation was conducted in Weiyuan, Gansu province, China, during cool, rainy weather. In 50% of the investigated fields (n = 10, the average size of fields was 0.14 ha), visible gray mold was observed on stems of A. membranaceus. In those fields, ∼30 to 50% of the stems were withered and necrotic, with abundant dark brown mold from the presence of conidiophores. To determine the causal agent of disease, 12 symptomatic stem samples were collected from four fields (three samples per field). The samples were disinfected with 10% sodium hypochlorite for 1 min, cut into pieces (2 to 3 × 10 mm, n = 12), rinsed three times with sterile water, and dried on sterile tissue. Samples were placed on potato dextrose agar (PDA), three pieces per plate, and were incubated at 25°C in the dark. After 3 days, hyphal tips growing from the disinfected tissues were individually transferred to new PDA plates and incubated at 25°C in the dark. From the 10 isolates obtained, GWT6-2 was chosen as a representative isolate for further study. Colonies of GWT6-2 had gray aerial mycelia with margins that eventually turned taupe. In addition, some black, hard sclerotia (1.93 to 12.21 × 1.29 to 11.57 mm, n = 20) with a round or irregular shape developed after ∼10 days of incubation at 25°C in the dark. Hyaline and round or elliptical conidia (7.86 to 14.53 × 6.81 to 11.79 μm, n = 50) were attached on top of brown conidiophores. Based on morphological characteristics, the isolate was initially identified as Botrytis sp. (Ellis 1971). For pathogenicity tests, 5 mm agar plugs from 1-week-old cultures of GWT6-2 were placed on four stabbed stems of A. membranaceus planted in 230 × 170 mm flowerpots. A similar number of plants inoculated with PDA plugs (5 mm) were used as a control, and the assay was replicated three times. The treatments were kept in plastic buckets with a temperature of 20 to 25°C and relative humidity of 80 to 90%. After 48 h, the agar plugs were removed, and the plants were kept in the buckets for 3 to 4 days. Waterlogged spots developed on the stems of A. membranaceus 3 days after inoculation, which elongated to 3 to 5 cm after 4 to 5 days of inoculation, and the gray mold that appeared on the diseased lesions resembled that observed in the fields. The pathogen was reisolated from diseased tissues, and the cultural (colonies) and microscopic characteristics (conidia and conidiophores) were similar to those of the original isolate. Controls were asymptomatic. To further identify the species, GWT6-2 genomic DNA was extracted, and the internal transcribed spacer (ITS), heat shock protein (HSP60), and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes were amplified with the primers ITS1/ITS4 (White et al. 1990), HSP60-F/HSP60-R, and G3PDH-F/G3PDH-R (Staats et al. 2005), respectively. The PCR amplicons were sequenced and compared with other sequences in GenBank using BLAST. The ITS, HSP60, and G3PDH sequences of GWT6-2 (MT225784.1, MT230538.1, MT263015.1) were 99.08, 100, and 99.25% identical to sequences of Botrytis cinerea strain GZFQ-1 (MH454037.1, MH479931.1, MH479930.1) and B. cinerea isolate 5-3 (MH718836.1, MH796663.1, MH796662.1), respectively. A multilocus phylogenetic tree was constructed with the ITS, HSP60, and G3PDH reference sequences, and the grouping of strain GWT6-2 was supported by 99% bootstrap value. Based on the morphological characteristics and molecular identification, the strain GWT6-2 was identified as B. cinerea. To our knowledge, this is the first report of B. cinerea causing gray mold on the stems of A. membranaceus in China. B. cinerea may affects the aerial growth of A. membranaceus, causing economic losses of this important medicinal plant. Therefore, further investigations of the impact of this pathogen on A. membranaceus growth and management options are needed.The author(s) declare no conflict of interest.References:Ellis, M. B. 1971. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, U.K. Google ScholarFu, J., et al. 2014. Phytother. Res. 28:1275. https://doi.org/10.1002/ptr.5188 Crossref, ISI, Google ScholarStaats, M., et al. 2005. Mol. Biol. Evol. 22:333. https://doi.org/10.1093/molbev/msi020 Crossref, ISI, Google ScholarWhite, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Google ScholarThe author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 106, No. 5 May 2022SubscribeISSN:0191-2917e-ISSN:1943-7692 Download Metrics Downloaded 562 times Article History Issue Date: 28 Apr 2022Published: 30 Mar 2022First Look: 23 Oct 2021Accepted: 20 Oct 2021 Page: 1520 Information© 2022 The American Phytopathological SocietyKeywordsAstragalus membranaceusBotrytis cinereagray moldThe author(s) declare no conflict of interest.PDF download

Tussilago farfara is of vital medical value. A new leaf spot disease was observed on T. farfara leaves, in Dingxi, Gansu Province, China, in October 2019. In order to research the pathogen, the diseased samples were collected for isolation and identification. The isolate KD3 was verified by pathogenicity test, as the pathogen causing the T. farfara leaf spot disease. Its morphological characteristics were consistent with Alternaria alternata, the colony color gray-green with concentric rings, conidia fusiform and pear-shaped, brown, with 1-7 septa and 0-3 lon-gitudinal septa, conidia size (19. 62-44.49) mu m x (6.97-10.53) mu m, beak length (1.35-10.03) mu m x (1.01-3.63) mu m, and the spore phenotype was a dwarf tree-like chain of short conidia. Multilocus sequences analysis man-ifested that the internal transcribed spacer (ITS), Alternaria major allergen (Alta1), and Calcium barine (CAL) sequences of strain KD3 were most closely to A. alternata (A23), with the homology of 99.47%, 99.56% and 98.28%, respectively. Based on morphological and molecular characteristics, strain KD3 was identified as A. alternata. OA was the optimal medium for its growth and PCA medium was the optimal for sporulation. This is the first report of A. alternata causing T. farfara leaf spots in China.

Potato soft rot, caused by the pathogenic bacterium Lelliottia amnigena (Enterobacter amnigenus), is a serious and widespread disease affecting global potato production. Both salicylic acid (SA) and proline (Pro) play important roles in enhancing potato tuber resistance to soft rot. However, the combined effects of SA and Pro on defense responses of potato tubers to L. amnigena infection remain unknown. Hence, the combined effects of SA and Pro in controlling newly emerging potato soft rot bacteria were investigated. Sterilized healthy potato tubers were pretreated with 1.5 mM SA and 2.0 mM Pro 24 h before an inoculation of 0.3 mL of L. amnigena suspension (3.69 × 107 CFU mL−1). Rotting was noticed on the surfaces of the hole where the L. amnigena suspension was inoculated. Application of SA and Pro with L. amnigena lowered the activity of pectinase, protease, pectin lyase, and cellulase by 64.3, 77.8, 66.4 and 84.1%, and decreased malondialdehyde and hydrogen peroxide contents by 77.2% and 83.8%, respectively, compared to the control. The activities of NADPH oxidase, superoxide dismutase, peroxide, catalase, polyphenol oxidase, phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, 4-coumaryl-CoA ligase and cinnamate-4-hydroxylase were increased in the potato tubers with combined treatments by 91.4, 92.4, 91.8, 93.5, 94.9, 91.3, 96.2, 94.7 and 97.7%, respectively, compared to untreated stressed tubers. Six defense-related genes, pathogenesis-related protein, tyrosine-protein kinase, Chitinase-like protein, phenylalanine ammonia-lyase, pathogenesis-related homeodomain protein, and serine protease inhibitor, were induced in SA + Pro treatment when compared with individual application of SA or Pro. This study indicates that the combined treatment of 1.5 mM SA and 2.0 mM Pro had a synergistic effect in controlling potato soft rot caused by a newly emerging bacterium.

Astragalus membranaceus, known as Huang-qi, is a perennial herbal medicine plant that grows 50 to 150 cm high, with 13 to 31 small leaves (Fu et al. 2014). In September 2019, a disease investigation was conducted in Weiyuan, Gansu province, China, while the weather was rainy and cool. In 50% of the investigated fields (n=10, the average size of 10 fields is 0.14 ha), visible gray mold was observed on the stems of A. membranaceus. Approximately 30%-50% of the stems in those fields were withered and necrotic, with abundant dark brown mold due to presence of conidiophores. To further determine the causal agent of disease, 12 symptomatic samples were collected from 4 different fields (3 symptomatic samples per field). The stem samples were disinfected with 10% sodium hypochlorite for 1 minute, cut into pieces (2 to 3mm × 10 mm, n=12), rinsed 3 times with sterile water, and dried on sterile tissue. Samples were then placed on potato dextrose agar (PDA), 3 pieces per plate, which were incubated at 25℃ in the dark. After 3 days, hyphal tips growing from the disinfected tissues were individually transferred to new PDA plates and incubated at 25℃ in the dark. From the ten isolates obtained, GWT6-2 was chosen as a representative isolate for further study. Colonies of GWT6-2 had gray aerial mycelia with the margins that eventually turned taupe. In addition, some black and hard sclerotia (1.93 to 12.21 mm × 1.29 to 11.57 mm, n=20) with round or irregular shape developed on the colonies after approximately 10 days of incubation at 25℃ in the dark. Hyaline and round or elliptical conidia (7.86 to 14.53 μm × 6.81 to 11.79 μm, n=50) were attached on the top of brown conidiophores. Based on morphological characteristics, the isolate was initially identified as Botrytis sp. (Ellis 1971). For pathogenicity tests, 5 mm agar plugs prepared from 1-week-old cultures of GWT6-2 were placed on four stabbed stems of A. membranaceus (planted in flowerpots, 230 × 170 mm). A similar number of plants inoculated with PDA plugs (5 mm) were used as control, and the assay was replicated 3 times. All the treatments were kept in plastic buckets with a temperature of 20℃-25℃ and relative humidity of 80%-90%. After 48h, the agar plugs were removed, and the plants were maintained in the plastic buckets for 3-4 days. Waterlogged spots developed on the inoculated stems of A. membranaceus 3 days after inoculation, which elongated to 3-5cm after 4-5 days of inoculation, and the gray mold that appeared on the diseased lesions was similar to that observed in the fields. The pathogen was reisolated from diseased tissues, and the cultural (colonies) and microscopic characteristics (conidia and conidiophores) were similar to those of original isolate. However, controls were asymptomatic. To further identify the species, the genomic DNA of GWT6-2 was extracted, and the internal transcribed spacer (ITS), heat shock protein (HSP60) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes were amplified with the primers ITS1/ITS4 (White et al. 1990), HSP60-F/HSP60-R and G3PDH-F/G3PDH-R (Staats et al. 2005), respectively. The PCR amplicons were sequenced and compared to other sequences in GenBank using BLAST. The results indicated that the ITS, HSP60 and G3PDH sequences of GWT6-2 (MT225784.1, MT230538.1, MT263015.1) were 99.08%, 100% and 99.25% identical to the sequences of B. cinerea strain GZFQ-1 (MH454037.1, MH479931.1, MH479930.1) and B. cinerea isolate 5-3 (MH718836.1, MH796663.1, MH796662.1), respectively. A multilocus phylogenetic tree was constructed with the ITS, HSP60 and G3PDH reference sequences, and the result revealed that the grouping of strain GWT6-2 was supported by 99% bootstrap value. Based on the morphological characteristics and molecular identification, the strain GWT6-2 was eventually identified as Botrytis cinerea. To our knowledge, this is the first report of B. cinerea causing gray mold on the stems of A. membranaceus in China. The occurrence of B. cinerea may affects the aerial growth of A. membranaceus resulting in economic yield losses of this important medicinal plant. Therefore, further investigations into the impact of this pathogen on A. membranaceus growth and management options are needed.

Bacterial antagonists are effective as an alternative to synthetic bactericides in the control of potato soft rot. The use of bioagents reduces the application of synthetic bactericides, which are harmful to humans and the environment. However, the mechanisms of some bioagents, such as some fungi and bacteria, are not yet understood. This paper reviews the current situation of potato soft rot, biological controls, antagonistic bioagents and their mechanisms, application strategies and future directions in today's agriculture. These mechanisms include mycoparasitism, competition, rhizosphere colonisation, synthesis and release of metabolites. Bioagents increased the defensive system of plants by increasing the antioxidants genes, such as superoxide dismutase, peroxidase (POD) and catalase (CAT), and eventually increased the plant growth and yield production.