Bipolaris australiensis HD-1胆红素氧化酶纯化、鉴定及其酶学性质

[目的]为了获得B.bipolaris HD-1胆红素氧化酶纯品,确定酶的同源性及其常规酶学性质.[方法]将B.bipolaris HD-1发酵液依次通过硫酸铵盐析、阴离子交换层析和凝胶过滤层析确定蛋白的纯度、浓度和酶活力;通过基质辅助激光解析电离飞行时间质谱（MALDI-TOF）法获得氨基酸序列,并利用Mascot软件在NCBI蛋白库中的比对,以确定同源蛋白来源;用SDS-PAGE方法测其表观分子量;用等电聚焦方法测定等电点;用Linwerver-Burk双倒数作图法测定米氏常数;并对该酶的酶学性质进行了常规的测定.[结果]获得具有胆红素氧化酶酶活性的电泳纯单体蛋白,酶活为46 000 U/L,比活为1.15 U/mg;MALDI-TOF共获得135个氨基酸,该蛋白与来源于Hel-minthosporium tritici-vulgaris的漆酶前体蛋白序列同源性达100％;蛋白的表观分子量为68 kDa;pI为4.1;它对ABTS和胆红素的米氏常数分别为Km（ABTS） =1.8×10-5 mol/L（pH 3.0）和Km（bilirnbin）=2.7×10-5 mol/L（pH 7.5）;对胆红素的最适催化活性为36℃,酶在-40℃下可保持2年,酶活保持在90％以上,在pH 8 ～9.5下可保持相当高的催化胆红素活性,能够耐受高浓度的硝酸钠和尿素,低浓度的叠氮化钠对酶活有促进作用而高浓度则抑制,对氯化钠、溶解氧敏感.[结论]该蛋白具有典型的胆红素氧化酶特性,又存在明显的不同.

Induced Resistance by the Toxin Filtrate of Bipolaris maydis Race T Cultivation

Resistance to maize southern leaf spot disease was induced by the low-concentration filtrate of Bipolaris maydis race T cultivation in an experiment. The nuclear neterogeny corn C103 was used as the test material. The lesion area on the leaves was significant difference by connalysis. The lesion areas on pretreated leaves were （0.3±0.05）- （0.9±0.5） mm^2, but those on the control were （23.1±8.7） mm^2. At the same time, the changes in peroxidase, phenylalanine, ammonialyase, and malondialdehvde activities were determined. During 0-96 h of inspection, phenylalanine and ammonialyase （PAL） activities increased by 64.2%, peroxidase （POD） activities increased by 41.2%, but the malondialdenvde （MDA） content decreased by 29.7% compared with the control. It seems that the low-concentration filtrate of Bipolaris maydis race T cultivation itself can be used as an elicitor to enhance the induced resistance.

Functional Analysis of MYB73 of Arabidopsis thaliana Against Bipolaris oryzae

Bipolaris oryzae is a severe rice disease,resulting in significant economic losses.MYB73 gene of Arabidopsis was isolated based on T-DNA insertion mutants whose lose-of-function mutant increased susceptibility to B.oryzae.Results of staining for H2O2 revealed that infection of incompatible B.oryzae caused much more strong brown patches on the leaves of myb73 mutant than those on the leaves of wild type.Expression of MYB73 gene was induced by B.oryzae.Its increased expression was severely impaired in the myb73 mutant.Expression of MYB73 was severely decreased in the npr1,jar1,eds5,and sid2 mutants,suggesting MYB73 gene participates in the jasmonate （JA） and salicylic acid （SA） signaling pathways.Expression of MYB73 who encoded an R2R3 MYB transcription factor was increased by SA,JA,and ethylene （ET） treatments.The cDNA full-length sequence of MYB73 was 960 bp and a protein with 320 amino acids was encoded.The predicted molecular weight of MYB73 was 34.85 kDa.The expression of MYB73 gene was induced within 24 h of the inoculation,however,the expression of PR1,PDF1.2 and NPR1 weren＇t changed.When B.oryzae successfully infected myb73 mutant plants,the expression of PR1,PDF1.2 and NPR1 were increased.Collectively,our results suggested that MYB73 is involved in NPR1-mediated SA and JA signaling pathways.

The antifungal properties of terpenoids from the endophytic fungus Bipolaris eleusines

A series of terpenoids(1-17),comprising six new compounds designated bipolariterpenes A-F(1-6)and eleven rec-ognized compounds(7-17),were isolated from the wheat culture of the potato endophytic fungus Bipolaris eleusines.Their structures and stereochemistry were clarified by HRESIMS,NMR,DP4+probability analyses,and computations for electronic circular dichroism(ECD).All compounds are made up of six meroterpenoids,four sesterterpenes and seven sesquiterpenes.Among them,four sesterterpenes(4,5,10,11)were investigated for their antifungal,antibacterial and cytotoxic properties,and six meroterpenoids(1-3,7-9)were evaluated for their antifungal properties.The compounds 7,9,and 10 had substantial antifungal activity against Epidermophyton floccosum at a concentration of 100μM.No antibacterial and cytotoxic activities were observed.

Screening and Identification of Biocontrol Strains against Bipolaris sorokiniana

Bipolaris sorokiniana is an important disease causing wheat root rot. Different biocontrol strains were screened with the pathogen as the target. A total of 210 strains of bacteria and fungi preserved in the laboratory were preliminarily and secondarily screened by plate confrontation culture. The 17 strains of bacteria and 36 strains of fungi with good bicontrol effect were screened. Eventually, the best fungal strain and the best bacterial strain were obtained. The ITS fragments of fungi were amplified by PCR, and the target band of 571 bp was obtained. The 16 S gene frag-ments of bacteria were amplified by PCR, and the target band of 1 455 bp was obtained. Through morphological observation, the screened strains were identified. Finally, the bicontrol fungus was confirmed as Trichoderma asperellum and the bacterium was Bacillus amylofaciens.

Seco-sativene and Seco-longifolene Sesquiterpenoids from Cultures of Endophytic Fungus Bipolaris eleusines

Two new seco-sativene sesquiterpenoids,bipolenins D(1)and E(2),a new seco-longifolene sesquiterpenoid,bipolenin F(3),together with three known analogues(4–6),were obtained from cultures of endophytic fungus Bipolaris eleusines.Their structures were established by MS and NMR data.Compounds 1–6 showed no activity to five human cancer cell lines.

Identification and Expression Analysis of Regulatory Genes Induced by Near-Ultraviolet Irradiation in <i>Bipolaris oryzae</i>

Bipolaris oryzae is the causal agent of brown leaf spot disease in rice, and its asexual spore (conidium) formation is known to be induced by near-ultraviolet (NUV) irradiation. In order to reveal the photomorphogenic response and to identify new genes upregulated by NUV irradiation, suppression subtractive hybridization (SSH) was carried out in B. oryzae. To confirm the differential gene expression in NUV-irradiated mycelia, quantitative real-time PCR (qRT-PCR) analysis was performed among 301 genes arbitrarily chosen from 1170 cDNA clones. The expression of 46 genes (named NUV01 to NUV46) was found to be significantly enhanced (>4-fold) by NUV irradiation. Sequence analysis revealed that 23 out of the 46 sequences (50%) showed significant matches to known fungal genes. The 46 genes were categorized as either BLR1-dependent or BLR1-independent expression groups using the BLR1-deficient mutant, which presumably lacks the blue/UVA-absorbing photoreceptor. This finding demonstrates that NUV irradiation can induce gene regulation, and that this response may be mediated by both a blue/UVA-absorbing photoreceptor and an as-yet-unidentified photoreceptor in B. oryzae.

Expression of <i>T4HR1</i>, a 1,3,6,8-Tetrahydroxynaphthalene Reductase Gene Involved in Melanin Biosynthesis, Is Enhanced by Near-Ultraviolet Irradiation in <i>Bipolaris oryzae</i>

Bipolaris oryzae is the causal agent of brown spot disease in rice and produces the dark pigment melanin. We isolated and characterized T4HR1 gene encoding 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN) reductase, which converted 1,3,6,8-THN to scytalone in the melanin biosynthesis from B. oryzae. A sequence analysis showed that the T4HR1 gene encoded a putative protein of 268 amino acids showing 50% - 99% sequence identity to other fungal 1,3,6,8-THN reductases. Targeted disruption of the T4HR1 gene showed a different phenotype of mycelial color due to an accumulation of shunt products compared to those of wild-type on PDA plates using tricyclazole as a melanin biosynthesis inhibitor. A quantitative real-time PCR analysis showed that the expression of T4HR1 transcripts was enhanced by near-ultraviolet (NUV) irradiation and regulated by transcriptional factor BMR1, similar to three other melanin biosynthesis genes (polyketide synthase gene [PKS1], scytalone dehydratase gene [SCD1], and 1,3,8-THN reductase gene [THR1]) in the melanin biosynthesis of B. oryzae. These results suggested that common transcriptional mechanisms could regulate the enhanced gene expression of these melanin biosynthesis genes by NUV irradiation in B. oryzae.

Efficacy of Plant Extracts on Morphology and Cultural Characteristics of Bipolaris sorokiniana, Causing Black Point Disease of Wheat in Bangladesh

Black point of wheat caused by Bipolaris sorokiniana (Bs) is very destructive disease of wheat in Bangladesh and all over the world. The symptoms of diseases caused by Bipolaris sorokiniana can vary depending on the host plant and environmental conditions. The conidia of Bs germinate from two poles, which differ physiologically as indicated by the rate, growth character, of length, breadth and number of septa on different media. The aim of this study was to evaluate the efficacy of selected plant extracts on the morphology and cultural characteristics of the fungus Bipolaris sorokiniana causing black point disease in wheat crops in Bangladesh. The efficacy of fifteen plant extracts on the growth and characteristics of Bs was conducted in vitro in 2019-2020. The treatments were: Untreated control, T1—Black berry Leaves, T2—Guava Leaves, T3—Lantena camera leaves, T4—Eucalyptus Leaves, T5— Turmeric Leaves, T6—Khoksha Leaves, T7—Papaya leaves, T8-Gurlic Bulb, T9—Chili dust, T10—Nigella seeds, T11—Turmeric dust, T12—Cloves, T13— Bohera fruits, T14—Black pepper and T15—Neem leaves. After 15 days of inoculation of Bipolaris sorokiniana, colony color and shapes were Gerrish blackish, greenish blackish, greyish blackish and shapes were round or irregular. In our study, conidia color was dark brown to light brown, conidiophore color was brown, dark brown, grayish brown, dark olivaceous, light brown and conidia shapes were elliptical, oblong or slightly curved. The septation of conidia was 2 - 5 and highest septation was seen in T4 and T14 treatments. The highest conidial length was 17.79 μm recorded in T2 treatment and lowest was 6.62 μm T9 treatment where conidial breath was 8.27 μm in control and lowest was 3.79 μm in T8 treatment. Mycelial growth rate of Bipolaris sorokiniana was different in different treatments in different days after inoculation (DAI) where at 7 DAI, % reduction of mycelial growth over control was highest in Bohera and Neem Leaves treatments respectively.

Hordedane diterpenoid phytoalexins restrict Fusarium graminearum infection but enhance Bipolaris sorokiniana colonization of barley roots

Plant immunity is a multilayered process that includes recognition of patterns or effectors from pathogens to elicit defense responses.These include the induction of a cocktail of defense metabolites that typically restrict pathogen virulence.Here,we investigate the interaction between barley roots and the fungal pathogens Bipolaris sorokiniana(Bs)and Fusarium graminearum(Fg)at the metabolite level.We identify hordedanes,a previously undescribed set of labdane-related diterpenoids with antimicrobial properties,as critical players in these interactions.Infection of barley roots by Bs and Fg elicits hordedane synthesis from a 60o-kb gene cluster.Heterologous reconstruction of the biosynthesis pathway in yeast and Nicotiana benthamiana produced several hordedanes,including one of the most functionally decorated products 19-β-hydroxy-hordetrienoic acid(19-OH-HTA).Barley mutants in the diterpene synthase genes of this cluster are unable to produce hordedanes but,unexpectedly,show reduced Bs colonization.By contrast,colonization by Fusarium graminearum,another fungal pathogen of barley and wheat,is 4-fold higher in the mutants completely lacking hordedanes.Accordingly,19-OH-HTA enhances both germination and growth of Bs,whereas it inhibits other pathogenic fungi,including Fg.Analysis of microscopy and transcriptomics data suggest that hordedanes delay the necrotrophic phase of Bs.Taken together,these results show that adapted pathogens such as Bs can subvert plant metabolic defenses to facilitate root colonization.

cDNA-AFLP analysis reveals that maize resistance to Bipolaris maydis is associated with the induction of multiple defense-related genes

The fungal pathogen Bipolaris maydis invades by direct penetration into maize leaf veins. In order to understand the resistance mechanism of maize to B. maydis strain 523, cDNA-AFLP (amplified fragment length polymorphism) analysis was conducted to compare the changes in mRNA transcripts hi response to B. maydis infection between a highly disease-resistant (HDR) line and a susceptible (S) line. 13 cDNA fragments derived from the genes showing enhanced expression after fungal infection, named HDR genes, were isolated from the HDR line. Northern blot analysis showed that 5 HDR genes were induced by fungal infection in the HDR, but not the S lines. The 5 HDR genes showed homology to previously characterized genes involved in disease resistance. A full-length HDR10 cDNA was isolated. It had a capacity to encode a protein of 284 amino acids. The deduced amino acid sequence of the HDR10 gene was homologous to a fungal infection-induced protein from Cicer arietinum and a hypersensitive response protein from

Bipolarins A–H, eight new ophiobolin-type sesterterpenes with antimicrobial activity from fungus Bipolaris sp. TJ403-B1

Bipolarins A–H(1–8), eight new tetracyclic ophiobolin-type sesterterpenes featuring a rare oxaspiro[4.4]nonane moiety, were isolated from cultures of fungus Bipolaris sp. TJ403-B1. Their structures and absolute configurations were elucidated by comprehensive spectroscopic analyses, single-crystal X-ray diffraction experiments, electronic circular dichroism and 13 C NMR calculations. Additionally, compound 5 exhibited significant selective antimicrobial activity against Enterococcus faecalis with an MIC value 8 μg·mL^-1.

洋金花内生真菌Bipolaris sp.的次级代谢产物研究

目的对洋金花内生真菌Bipolaris sp.的次级代谢产物进行研究。方法采用多种色谱法对洋金花内生真菌Bipolaris sp.的次级代谢物进行分离,并结合理化性质及核磁共振波谱解析等方法进行结构鉴定。采用MTT法测定化合物对肺癌细胞株A549和肝癌细胞株Hep G2的细胞毒性。结果与结论从洋金花内生真菌Bipolaris sp.的次级代谢物中分离鉴定出10个化合物,分别为helminthosporol(1)、9-hydroxyhelminthosporol(2)、helminthosporic acid(3)、helminthosporal acid(4)、cochliobolin A(5)、prehelminthosporol(6)、fusaproliferin(7)、(-)-terpestacin(8)、isocochlioquinone B(9)和isocochlioquinone C(10),其中化合物4、5、9为首次从该菌次级代谢物中分离得到。化合物2、4、5、7~9对肺癌细胞株A549和肝癌细胞株Hep G2具有细胞毒性(IC50≤50μmol·L^(-1))。

The pathogenic site of the C-toxin derived from Bipolaris maydis race C in maize (Zea mays)

Bipolaris maydis race C strain 523 (C523) induces severer leaf blight on cytoplasmic male sterility (CMS)-C maize than on normal (N) maize. Previously, a pathotoxin isolated from C523 (C-toxin) was shown to be responsible for the disease. To understand the basis of the differential responses between CMS-C and N maizes to this fungus, protein synthesis in vitro by mitochondria from N and CMS-C cytoplasms was monitored after their incubation in a solution containing the toxin (0.3%). Similar protein products were detected between the two alloplasmic lines, indicating that the toxin does not directly act on the mitochondrial membrane, nor inhibits the expression of mitochondrial genes. To further locate the action site of the toxin, intact leaves from both N and several subtypes of CMS-C lines were treated by 0.3% toxin. Analysis of electrolyte leakage of leaf cells showed that the leakage rates were similar to one another among the alloplasmic maize lines. In contrast, at a lower concentration of the

Meroterpenoids with Potent Immunosuppressive Activity from Fungus Bipolaris zeicola

Six new meroterpe no ids,bipolarinoids A-F(1-6),and two known congen ers(7-8)were isolated from a soil-derived fungus Bipolaris zeicola.Their structures were elucidated based on extensive spectroscopic techniques(ineluding HRESIMS and ID and 2D NMR data),and the absolute configurations of 1-4 were further confirmed viasingle-crystal X-ray diffraction an alysis and comparis on of experimental electronic circular dichroism(ECD)data.Structurally,compound 1 represents the first case of cochlioquinone derivatives featuring a formaldehyde group at C-11, and compou nd 3 features a rareamino group at C-11.In the immunosuppressive activity assay,compounds 5-8 showed potent inhibitory activity against concanavalin A(ConA)-induced T lymphocyte proliferation with IC50 values of 78.3,81.1,48.3,and 48.0 nmol/L,respectively,which provided promising leads for designing and developing new im-muno suppressive agents to treat auto-immunological diseases.

河南省玉米小斑病菌生理小种鉴定及致病力分化

2013年从河南省4个地区采集玉米小斑病标样,对分离出的140个玉米小斑病菌菌株进行了生理小种鉴定和群体结构分析。结果表明,河南省玉米小斑病菌有T、C、S、O等4种生理小种,但各小种在地区间的分布存在差异,其中,O小种是河南省各玉米产区小斑病菌的优势小种,且在豫南分布最广泛,其次是C小种和S小种,T小种的比例最小。O小种具有不同致病力,强致病力菌株的出现频率较弱致病力菌株高,强致病力菌株主要分布在豫南地区,弱致病力菌株主要分布在豫北地区。

重庆地区玉米圆斑病菌生物学特性的测定

试验从温度、pH值、光照、碳氮源的利用等方面对玉米圆斑病病原菌的生物学特性进行了较为系统的研究.结果表明：玉米圆斑病菌菌丝在5-40℃之间均能生长,而在10-35℃之间能产孢,最适生长和产孢温度为25℃,菌落直径达62 mm,产孢量为1.04×105（个/mL）;病原菌在pH 2-12之间均能生长和产孢,其中在pH为7时病原菌菌丝生长最好,菌落直径达35.3 mm,pH为8时病原菌产孢量最大,为2.06×105（个/mL）;光暗交替有利于病原菌产孢,光照对菌丝的扩展基本没影响;该病原菌对单糖、双糖、多糖等碳源以及有机氮和无机氮均能利用,其中以乳糖作碳源、甘氨酸作氮源时菌丝生长最佳,以D-木糖作碳源、以牛肉膏作氮源时产孢最好.

1株产胆红素氧化酶新菌株的分离与鉴定研究

[目的]分离与鉴定1株产胆红素氧化酶的新菌株。[方法]利用常规的微生物分离培养技术,从玉米叶片的病斑中分离出一株产胆红素氧化酶(bilirubin oxidase,BOD)的丝状真菌。[结果]通过形态学、生理学和rDNA-ITS(登录号为JN251106)序列分析,该菌株被鉴定为Bipolaris australiensis,命名为B.australiensis HD-1,属子囊菌门格孢腔菌目格孢腔菌科平脐蠕孢属澳大利亚平脐蠕孢霉(B.australiensis),亦称澳洲双孢霉,是1株没有被报道过的具有产BOD的新产生菌;在不含任何诱导剂的发酵液中28℃,150 r/min,培养6 d,其酶活可达为1 000 U/L。[结论]该方法成功分离出1株产胆红素氧化酶的新菌株,为BOD的产生菌提供了新的微生物来源。

小麦黑胚病病原菌的分离及致病力差异的初步研究

对供试的6个小麦品种进行小麦黑胚病调查统计。结果显示,6个品种均有小麦黑胚病发生,前后两代籽粒黑胚率分别为0.10%～21.10%和0.26%～25.22%,且不同品种间的发病程度存在差异。根据柯赫氏法则,分离所得主要病原菌为突脐蠕孢(Bipolaris sorokiniana)和细链格孢(Alternaria alternata),分离率分别为6.67%和76.67%;通过温室滴接试验,两种菌均能引起小麦黑胚病,突脐蠕孢和细链格孢的回接分离率分别为99.50%和96.67%,前者的致病力比后者强;小麦黑胚病的两种主要病原菌从扬花期至乳熟期均可侵染,以扬花盛期和灌浆初期为侵染的最适时期。

Identification of Antifungal Substance (Iturin A_2) Produced by Bacillus subtilis B47 and Its Effect on Southern Corn Leaf Blight

Bacillus subtilis B47 is an endophytic bacterium of tomato, and produces substance that strongly inhibits the growth of Bipolaris maydis, the pathogen of southern corn leaf blight （SCLB）, as well as several other phytopathogenic fungi. The antifungal substance was purified from the broth culture of the bacterium using acid precipitation, methanol extraction, and three-step chromatography. Based on FT-IR spectrometry, amino acid composition, and MALDI-TOF-MS/MS CID analyses, the antifungal substance was identified as iturin A2, a cyclic lipopeptide antibiotic. To evaluate the efficacy of iturin A2 for control of SCLB, partially purified iturin A2 （75%, w/v） was applied under different conditions. At the concentration of 300 mg kg-1, iturin A2 showed efficacy ranging from 100 to 53.1% under in vitro, in plot and in field conditions. This efficacy was higher than or similar to that of the fungicide chlorothalonil. When the concentration of iturin A2 was increased to 500 mg kg-1, the control efficacy was enhanced to 64.2% in field, which was significantly higher than that of chlorothalonil. These results indicate that iturin A2 has potential for SCLB control and could be a substitute to synthetic fungicides. To our knowledge, this is the first report on using partially purified iturin A for control of SCLB under field conditions.