Molecular Characterization,Morphology and Pathogenicity of Curvularia pseudobrachyspora,A New Causal Agent of Leaf Spot on Banana

[Objectives]The paper was to study molecular characterization,morphology and pathogenicity of Curvularia pseudobrachyspora,a new causal agent of leaf spot on banana.[Methods]Banana(Musa acuminate)leaves with streaks or long ellipse-shaped lesions were sampled in an orchard of Danzhou City,Hainan Province,China in 2021.Fungus isolates were isolated from the diseased tissues and further identified as C.pseudobrachyspora based on morphological characteristics of colony,conidiophore and spore,phylogenetic analyses of the ITS region,GAPDH and TEF-1αgenes.[Results]In the pathogenicity test,the fungus re-isolated from inoculated leaves with necrotic lesions was identified morphologically and molecularly,fulfilling Koch's postulates.[Conclusions]C.pseudobrachyspora is a new pathogen causing leaf spot of banana in China and the world.

A new Curvularia lunata variety discovered in Huanghuaihai Region in China

The purpose of this study was to identify the dominant pathogens of Curvularia leaf spot and their pathogenicity variation in Huanghuaihai Region of China in recent years.In 2013 and 2016–2017,the occurrences of Curvularia leaf spots on maize were investigated in fields located in Henan,Hebei,Shandong,and Anhui provinces,and 292 fungi were isolated from diseased leaves.These fungal isolates were subjected to morphological identification,and 232 isolates were found to have about 70%uncurved conidia and were identified as Curvularia lunata var.Most of the conidia of 2 representative isolates,namely,HNWB-131 and HNWB-185,were oblong with parallel septations and were distinctly different from a reference isolate CX-3.For further determination,the internal transcribed spacer(ITS),glyceraldehyde 3-phosphate dehydrogenase(GPDH),the large subunit(LSU),and translation elongation factor 1-alpha(EF1-α)sequences of HNWB-131,HNWB-185,and CX-3 were amplified and sequenced.The results of sequence analysis showed that the 4 gene sequences from the 3 isolates had a similarity of more than 99%to C.lunata.Based on the sequences of ITS and the combined data of the 4 genes,neighbor-joining trees were constructed for phylogenetic analysis.The results indicated that these 3 isolates were clustered together with C.lunata.The expression of Clg2 p and ClUrase genes in mycelia and conidia was significantly(P<0.05)higher in CX-3 than in HNWB-131 and HNWB-185.This study found that the dominant pathogen of Curvularia leaf spot was a new variety of C.lunata with morphological variations in Huanghuaihai Region from 2013 to 2017.The pathogenicity of the C.lunata var.was not significantly enhanced,and the expression of Clg2 p and ClUrase genes of C.lunata var.was decreased.

玉米新月弯孢菌(Curvularia lunata)的RAPD分析

对分离自玉米弯孢菌叶斑病标样中的77株新月弯孢菌和1株来自水稻的新月弯孢菌进行RAPD分析表明,菌株间具有丰富的遗传多样性,在相似系数约0．60处,所有菌株被聚为3个组,但88．0％的菌株聚入第Ⅰ组内,其余菌株被聚入另外2个组内。第Ⅰ组内共有69个菌株,包含来自不同区域的致病性较强的菌株,是玉米弯孢菌叶斑病的优势类群,其余 2个类群主要是一些致病能力较弱或不致病的菌株。结果表明,新月弯孢菌种内菌株的遗传多样性与致病性相关,但与菌株地理来源无明显的直接关系。

玉米黄斑病研究──Ⅲ.病原菌Curvularia lunata致病毒素初探

用玉米黄斑病菌Curvularialunata无菌培养滤渡进行生物测定的结果表明，无菌培养滤波处理的，离休叶圆片四周有较大范围的褪绿区；活体植株叶片上产生水清状褪绿晕斑，与病原菌所致的症状相同；种子根的伸长受到明显抑制，证明C．lunata在人工培养过程中可以产生致病毒素。这种毒素是对热稳定的物质。关于C．lunata致病毒素尚属首次报道，作者将其命名为C1毒素。

转DsRed荧光蛋白的新月弯孢Curvularia lunata菌株构建

本研究通过农杆菌介导转化方法(ATMT),利用DsRed荧光蛋白基因对玉米弯孢叶斑病致病菌新月弯孢进行遗传转化。通过转化子的荧光蛋白基因和潮霉素B抗性基因的PCR检测,菌丝体和分生孢子的荧光观察,hyg基因的Southern杂交验证,以及荧光蛋白基因插入位点的TAIL-PCR分析,确定了DsRed荧光蛋白基因插入与表达对新月弯孢转化子的影响。结果表明:测定的4株转化子基因组中均成功整合了DsRed荧光蛋白目的基因片段;转化子在生长发育和致病性方面与野生型菌株存在一定差异,分别有2株在产孢量方面略高于野生型菌株,4株转化子在纤维素酶活性和粗毒素致病力方面均低于野生型菌株,有3株转化子在果胶酶活性上较野生型菌株有提高,1株转化子的致病力显著低于野生型菌株;获得其中3个转化子插入位点的侧翼序列。

温郁金弯孢霉叶枯病病原菌(Curvularia clavata)生物学特性研究

本文对温郁金叶枯病病原菌(Curvulariaclavata)生物学特性进行了研究,以期为温郁金叶病害的综合防治提供依据。结果表明,温郁金弯孢霉叶枯病病原菌生长的最适培养基是CA培养基,最适温度是30℃,最适pH值为7。不同碳源对弯孢霉菌丝生长影响差别不大,适宜碳源为葡萄糖,乳糖和D-山梨醇。不同氮源对弯孢霉菌丝生长有差异,适宜氮源为牛肉浸膏和酵母浸膏,弯孢霉菌在硫酸铵中生长缓慢。

稗弯孢菌(Curvularia Iunata)原生质体的制备

研究了不同菌龄、酶浓度、渗透压稳定剂、缓冲液pH值以及酶解温度和时间等因素对稗弯孢菌(Curvularia lunata)原生质体形成的影响。将培养18h的菌丝,以0.7mol/L KCl作为渗透压稳定剂,30℃下,经过2％溶壁酶和4％纤维素酶混合酶液(pH5.8)酶解4h,原生质体在静止条件下最大释放量达到 1.7×10~6/mL,再生率为0.76％。

Evaluation of Curvularia lunata Strain B6 as a Potential Mycoherbicide to Control Barnydrgrass (Echinochloa crus-galli)

The virulent strain B6 of Curvularia lunata was screened out from 65 fungus strains isolated from the diseased leaves of barnyardgrass （Echinochloa crus-galli）. Greenhouse and field studies were conducted to evaluate the feasibility of the strain being exploited as a mycoherbicide for barnyardgrass control in paddy fields. The results of pathogenicity experiments showed that this strain was highly pathogenic to barnyardgrass at the 1- to 2.5-leaf stages. The fresh weight reduction increased with the increase of inoculated conidial concentrations and the prolongation of dew duration. Strain B6 provided excellent barnyardgrass control when it was applied at the concentration of 1×104-1×106 conidia mL-1 in paddy fields with water layer. This strain was very safe to rice and the most plant species except wheat, barley and corn. Findings of this study indicated that this strain could be a potential mycoherbicide for barnyardgrass control in paddy fields in the future.

MAPKs and acetyl-CoA are associated with Curvularia lunata pathogenicity and toxin production in maize

Mitogen-activated protein kinase （MAPK） cascades play an important role in extracellular signal transduction and are involved in the pathogenicity of fungal pathogens to host plants. In Curvularia lunata, the roles of two MAPK genes, Clkl and CIm 1, have already been studied. Clkl is involved in conidia formation and pathogenicity, and Clmf is closely related to pathogen cell wall formation and pathogenicity to maize leaves. In this study, a third C. lunata MAPK gene, Clhl, which is homologous to hog1, was successfully cloned. We found that a Clhl deletion mutant had lower intracellular glycerol accumulation than the wild-type stain and was unable to grow normally under osmotic stress conditions. Furthermore, the deletion mutants of three C. lunata MAPK genes （Clkl, Clml and Clhl） had lower levels of acetyI-CoA, which is an important intermediate product in the synthesis of melanin and furan toxin, and down-regulated expression of pathogenicity-associated genes. Furthermore, pathogenicity and the ability to produce toxin were restored after adding acetyI-CoA to the culture medium, suggesting that acetyI-CoA is closely involved in the pathogen MAPK signaling pathway.

Curvularia lunata侵染不同抗性玉米品系的应答差异蛋白解析

用山东莱西市玉米弯孢叶斑病菌强致病类型菌株QN-10,接种玉米抗病自交系齐319和感病自交系7922,采用蛋白质组技术分析其差异蛋白及功能。结果表明,齐319感染弯孢菌3 d后,有12个差异蛋白质斑点被诱导合成,8个为增量表达蛋白质斑点,2个为减量表达蛋白质斑点,2个为新产生蛋白质斑点;自交系7922有3个蛋白质斑点表达量增加,2个斑点表达量下降,但没有发现新的蛋白质合成。齐319差异蛋白质斑点鉴定表明,分别属于β-1,3-葡聚糖酶、F-box亮氨酸高度重复蛋白、锌指蛋白。

Sod gene of Curvularia lunata is associated with the virulence in maize leaf

Curvularia leaf spot, caused mainly by Curvularia lunata, is a widespread plant disease in China. In the recent years, di- rectional host selection by the pathogen, which likely results in the virulence differentiation in pathogens, is widely reported. Among the hallmarks potentially associated to pathogen variation in virulence, superoxide dismutase gene Sod has been found to be closely related to the enhancement of virulence. In the present study, the full-length of Sod was obtained via Blastn alignment against GenBank and the whole genome of C. lunata. In order to understand the role of Sod in the vir- ulence variation in C. lunata, targeted gene disruption was performed to construct Sod mutants. The cell wall degrading enzyme （CWDE） activities and toxin production of ASod were not distinctly different from wild-type strain CX-3 and its complon. However, at an early stage of infection, 3Sod virulence appeared to be lower than CX-3 and the complon, while at a later stage, its virulence gradually returned to the level of CX-3 and the complon. Furthermore, the melanin production of ASod was significantly reduced compared to CX-3 and the complon, suggesting that Sod gene influences the virulence by regulating melanin production at an early stage of infection but is not essential for pathogenicity. However, the disruption of Sod did not significantly affect the transcriptional expression of the melanin biosynthesis-associated genes, bml and scd. Therefore, we infer that Sod in C. lunata are involved, to some extent, with the virulence in maize leaf, but still needs further studies to have a clear understanding of its mechanism.

First Report of Curvularia pseudobrachyspora Causing Leaf Spots Disease on Coconut (Cocos nucifera L.) Seedlings in Ghana

Coconut (Cocos nucifera L.) is an important oilseed and a multipurpose perennial plantation crop. It is mostly grown in humid and coastal areas of Ghana. In June 2019, leaf spot disease was observed on coconut seedlings in 10 coconut growing Districts in Ghana. The Initial symptoms appeared as elliptical, brown-dark-red lesions, 4 - 6 mm on affected leaves. Lesions reached 2.0 cm and gradually developed into spindly, dark brown spots with a light tan centre. A study including survey and laboratory work was carried out to assess disease prevalence and to identify the causal agent of the disease on coconut seedlings, in order to formulate effective management strategies against it. A total of 250 symptomatic leaves were picked from ten selected Districts for laboratory analysis. Additionally, the ribosomal internal transcribed spacer (ITS) region and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of the isolated pathogen were amplified using ITS1 and ITS4, gpd1 and gpd2 primers respectively. The disease incidence peaked at 95%. Fungal colonies on PDA grew to 50 - 70 mm in diameter in one week at a temperature of 28&deg;C ± 1&deg;C with an even to undulating, immersed striated, mycelium;aerial mycelium being dark velvety green and sometime woolly-cottony. Acervuli formed on the aerial mycelium and contained black powderish conidial masses. Hyphae characteristics were similar to that of Curvularia pseudobrachyspora. Pathogenicity tests were done following Koch’s postulate. For molecular confirmation, the combined ITS (MT075719) and GAPDH (MT075720) sequences were compared with published sequences of 52 Curvularia isolates and eight Bipolaris isolates using phylogenetic analysis. This is the first report of C. pseudobrachyspora as a causal agent of leaf spot on coconut seedlings in Ghana and possibly Africa but the pathogen has been reported on other crops in China, Florida and India.

Morphological and Molecular Identification of Curvularia geniculata Associated with Leaf Spot Disease of Banana in China

[Objectives]The paper was to identify the causal agent of leaf spot of banana in Hainnan,China.[Methods]Fungus isolates were isolated from the diseased tissues,and morphological characteristics of colony and spore were observed.Furthermore,phylogenetic analyses and pathogenicity test were conducted to confirm the pathogen.[Results]The fungus isolates from the diseased tissues was identified as Curvularia geniculata.[Conclusions]C.geniculata is a new pathogen causing leaf spot of banana in Hainan,China.This finding will help to broaden the distribution and host range of C.geniculata,indicating that it poses potential damage to banana in China.

Curvularin and Dehydrocurvularin as Phytotoxic Constituents from Curvularia intermedia Infecting Pandanus amaryllifolius

Microbes are good sources of biologically active compounds that can be used as pharmaceuticals and agrochemicals. As part of our continuous efforts in search for biopesticides from natural sources, a fungus is isolated from leaves of Pandanus amaryllifolius that shows severe necrosis. This fungus is cultured in potato dextrose agar and identified as Curvularia intermedia. The ethyl acetate extract of the liquid culture medium is phytotoxic. Identification and bioassay of αβ-dehydrocurvularin (1) and curvularin (2) from this fungus are described. Monoacetyldehydrocurvularin (3), and diacetyldehydrocurvularin (4) are synthesized from 1. Phytoxicity rankings in a variety of bioassays are 1 > 2 > 4 > 3.

Curvularia martyniicola,a new species of foliicolous hyphomycetes on Martynia annua from India

In the micromycofloristic survey of some dematiaceous hyphomycetes from the Terai region of Uttar Pradesh(India),an undescribed species(C.martyniicola)of anamorphic fungus Curvularia Boedijn was found on living leaves of Martynia annua(Martyniaceae).The novel fungus is described,illustrated and discussed in details.The present species is compared with earlier reported similar taxon,and is characterized by longer conidiophores and conidia with less septa.A key is provided to all the species of Curvularia recorded on Martyniaceae and Pedaliaceae.The details of nomenclatural novelties were deposited in MycoBank(www.MycoBank.org).

Analysis of the Protein Electrophoresis Patterns of Curvularia Lunata in Maize and Immunological Detection of the Pathogenic Culture

Twelve isolates of Curvularia lunata and six related species from maize or other host plants are examined for the analysis of polyacrylamide gel electrophoresis(SDS PAGE) patterns of their soluble proteins. Differences exist significantly among different species and the same species. There is a special protein band for Curvularia at relative mobility value 0.177 (Rf 0.177) and a special protein band for C. lunata at Rf 0.225. Two kinds of primary antibodies to C. lunata are used to indirect ELISA and Avidin Biotin peroxidase Complex (ABC) immunoassay to evaluate their detecting sensitivity and specificity. The sensitivity of ABC immuno assay is higher than indirect ELISA. APAbs to C. lunata is special to C. lunata.

Vercytochalasins A and B: Two unprecedented biosynthetically related cytochalasins from the deep-sea-sourced endozoic fungus Curvularia verruculosa

Vercytochalasins A(1) and B(2), two biosynthetically related cytochalasins featuring novel structure and substituents, were isolated from the endozoic fungus Curvularia verruculosa which was associated with the deep-sea squat lobster Shinkaia crosnieri collected from the cold seep environment in South China sea. Their structures were elucidated by detailed interpretation of NMR spectroscopic and mass spectrometric data. The absolute configurations were confirmed by NOESY experiments as well as by DP4+ and ECD calculations. Differed from common cytochalasins, compound 1 is an uncommon secocytochalasin featuring the ester group cleaved between C-9 and C-23, and incorporating an additional oxygenated C4 unit which coupled with C-20 and C-22 to form a new substituted cyclohexenone moiety, while compound 2 contains an unusual 2–hydroxy-3-oxobutan-2-yl unit at C-22. Both compounds are distinctive from the commonly described cytochalasins. Compound 1 exhibited potent activity against angiotensinI-converting enzyme(ACE) whereas compound 2 showed antibacterial activity. Molecular docking simulations were performed to explore the intermolecular interaction of compounds 1 and 2 with ACE.

Proteomics Associated with Virulence Differentiation of Curvularia lunata in Maize in China

One-dimensional electrophoresis （1-DE） of proteins, two-dimensional electrophoresis （2-DE） of proteins and cloning of cDNA sequence were used to study the virulence differentiation of Curvularia lunata （Wakker） Boed. isolated from maize （Zea maydis L.） in China. From 1-DE gel profiles of proteins, 110 reproducible bands were separated from six isolates of C. lunata CX-3, SD-6, C-152, C107-1, DD-60 and W-18. Sixty-eight bands （61.82%） were polymorphic, suggesting huge biodiversities among the isolates. All isolates for the experiment were clustered into three groups consisting of different virulent types by coefficient value of 0.605. Group 1, consisting of CX-3, SD-6 and C- 152 with high virulence displayed more protein bands than Groups 2 and 3, consisting of C107-1 and DD-60 with low virulence. Proteomics approaches based on 2-DE techniques were applied to identify specific proteins associated with the virulence differentiation in CX-3 and DD-60. A total of 423 protein spots were separated. Out of them 75 specific protein spots were displayed in 2-DE gels. Among them 28 protein spots were unique in CX-3 and eight in DD-60, and 39 protein spots were shown on both 2-DE gels but expressed differently in intensity. Twenty protein spots including three unique protein spots and 17 differentially expressed protein spots （more than two-fold DD- 60） in CX-3 were further identified with MALDI-TOF MS/MS. Results indicated that most of the identified proteins were found to be associated with virulence differentiation, metabolisms, stress response and signal transduction. One of them was identified as Brnl protein, which had been reported to be related to melanin biosynthesis and the virulence differentiation in fungi. Combined with our previous findings, we assumed that Brnl protein and its regulating products might be involved in the virulence differentiation of C. lunata. Consequently, we cloned a Brnl cDNA fragment and aligned it with the fragments in other fungi. Results indicated that the 633-bp sequence of Brnl cloned in C. lunata was highly homological with the compared fungi. Further work for the exact gene roles of Brnl in our case is underway.

西葫芦弯孢叶枯病病原菌Curvularia spicifera的鉴定

西葫芦(Cucurbita pepo L.)又名美洲南瓜,隶属葫芦科(Cucurbitaceae)南瓜属(Cucurbita spp.),原产于北美洲南部,世界范围内广泛种植^([1]),是促进我国农民增收的蔬菜作物之一^([2])。近年来,随着西葫芦保护地栽培面积的不断扩大,其病害发生普遍。生产上西葫芦细菌性病害和病毒病发生普遍^([1,3-4]),真菌性病害鲜见报道。2020—2021年,调查发现陕西省延安市南泥湾现代农业园区西葫芦叶枯症状发生严重.

欧洲种质对玉米主要叶部病害的抗性鉴定与评价

为筛选抗玉米叶斑病资源,应用人工接种方法对51份欧洲种质进行玉米大斑病、灰斑病、弯孢叶斑病的抗性鉴定与评价。结果表明,大部分种质表现为感病及高感,说明该批种质资源对以上3种叶斑病的抗性较差。筛选得到兼抗3种叶斑病的多抗性种质2份,抗性均达到中抗,为抗病品种的选育提供了重要材料。