Identification of mangrove endophytic fungus 1403 (Fusarium proliferatum) based on morphological and molecular evidence

A mangrove endophytic fungus 1403 isolated from the South China Sea Coast, which is able to produce griseofulvin and anthra- cenediones under submerged fermentation, was compared with Fusarium genus with the similar morphological characters such as elongated, microconidium-producing conidiophores, ovoid microconidia and straight to slightly curved macroconidia. It was found that the fungus 1403 resembles pathogenic E verticillioides （teleomophy Gibberella moniliforme） in the production of false head or chains and abundant microconidia on the aerial mycelium, but different in the occasional formation of polyphialides with relatively long as well as short mono- phialides, in its typical coiled hyphae and mycelia fusion. Through maximum Parsimony and Bayesian analyses, the fungus 1403 was fur- ther compared with some similar Fusarium species. The results indicated that this endophyte was identified as Fusarium proliferatum based on the analyses of partial 18S and 28S rDNA genes, ITS region, and EF- 1 α gene.

Isolation and identification of Fusarium oxysporum f. sp. cubense in Fujian Province, China

Fusarium wilt, caused by Fusarium oxyporum f. sp. cubense(Foc), is the most serious disease affecting banana production.To clarify the distribution of the Foc races in Fujian Province of China, 79 soil samples were collected from four regions of Zhangzhou City, the primary banana production area in Fujian. We isolated and identified 12 Foc strains based on internal transcribed spacer(ITS) sequence analysis, PCR amplification by using Foc-specific primers and pathogenicity assays.Our analysis indicated that 11 isolates belong to Foc race 1, and 1 isolate belongs to the Foc tropical species race 4(TR4).Although TR4 has previously been reported to occur in primary banana-producing provinces, such as Hainan, Guangxi,and Guangdong of China, this is the first report of TR4 isolated from the soil in Fujian Province. Monitoring the presence of Foc, in particular, the TR4 strains in the soil, is the basic strategy to prevent and control Fusarium wilt.

Isolation and Identification of Fusarium Wilt Pathogens from Bitter Gourd in Hainan Province

In order to isolate and identify the pathogens causing Fusarium wilt in bitter gourd in Hainan Province, China, four bitter gourd plants exhibiting symptoms of Fusarium wilt were collected from Tunchang County, Hainan Province. Four pathogen strains （HNTC-01, HNTC-02, HNTC-03 and HNTC-04） were isolated from the four plants, and identified via morphological observation and molecular techniques. The results showed that HNTC--01, HNTC-03 and HNTC-04 were F. oxysporum, while HNTC-02 was F. graminearum. The tests on pathogenicity and host specificity showed that HNTC-01, HNTC-03 and HNTC-04 were F. oxysporum Schl. f. sp. momordicae. Phylogenetic analysis revealed that HNTC-01, HNTC-03, HNTC-04 were clustered together with 14 strains of F. oxysporum, and HNTC-02 was clustered together with six strains of F. graminearum. In addition, rDNA-ITS region can be used to distinguish different Fusarium species, but can not be used to distinguish different biotypes within a Fusarium species. This study could provide a scientific basis for the diagnosis and prevention of wilt in bitter gourd.

Fusarium Wilt Resistance Identification and Genetic Analysis of Momordica charantia L. Germplasm

Fusarium wilt seriously affects the yield and quality of Momordica charantia L.Breeding resistant varieties is the most economical and effective measure to control Fusarium wilt in M.charantia L.This study had comprehensively conducted pathogen identification of Fusarium wilt,resistance evaluation of M.charantia L.germplasm to Fusarium wilt,and genetic analysis of Fusarium wilt resistance in M.charantia L.,so as to provide reference for breeding resistant varieties and accelerating the breeding process.

The morphological identification of Protoperidinium(Peridiniales, Dinophyceae) species on the coasts of China

The classification and identification for Protoperidinium species are the most difficult work during its taxonomic study. In this research, taxonomic status of Protoperidinium was clarified by tracing its taxonomic history, 23 species belong to genus Protoperidinium on the coasts of China were preliminarily identified, and morphological description and plate patterns were given for each species. The key differences of similar species were also discussed in this study, we believe that P. oceanicum and P. murry, P. tumidum and P. fatulipes, P. globules and P.majus are separate species; P. diabolum should be treated as the valid name instead of the reported names Peridinium globosum or Peridinium longipes; the taxonomic relationship between P. punctulatum and P.subinerme requires further study.

Vegetative Growth and Molecular Identification of Fusarium equiseti Isolated from Wilt Disease of Centella asiatica L. in Bangladesh

Centella asiatica (L.) is commonly known as Thankuni plant and has ethnobotanical importance in Bangladesh. Present experiment was conceded to investigate the wilt disease of C. asiatica, vegetative growth and molecular characterization of pathogenic fungi. Pathogenic fungus, Fusarium equiseti was identified as a causal agent of wilt disease in C. asiatica. The effect of culture media on the mycelial growth of F. equiseti showed the highest (89.25 mm) on potato dextrose agar (PDA) medium followed by carrot agar (CA) medium and the lowest growth (40.25 mm) was measured in HA medium. The optimal temperature and pH for mycelial growth of F. equiseti were 30°C and 7, respectively. The genetic variation of the selected species of fungi, the internal transcribed spacer (ITS) region was amplified using ITS4 and ITS5 primers and sequenced. The PCR product of the ITS region of F. equiseti was 535 bp. Phylogenetic tree of thirty-seven strains of Fusarium sp. based on the nucleotide sequences of the ITS region using the neighbor-joining method with 1000 bootstrapping indicated that 98% - 100% identity with MN886590.1 JUF0046 (F. equiseti). ITS sequences are generally constant, or show little variation within species, but vary between species in a genus. The ITS region is relatively short and can be easily amplified by PCR using universal single primer pairs. Genetic distance exhibited high level of similarity with identical ITS sequences. To date, no published research articles are found on the molecular identification of F. equiseti, the causal agent of fusarium wilt disease of C. asiatica in Bangladesh.

Identification of Fusarium Species Associated with Onion(Allium cepa L.)Plants in Field in Burkina Faso

Many fungi limit onion production in Burkina Faso. This study aims to identify the main Fusarium species associated with onion plant in field in order to determine those involved in seedling damping-off and bulb rot, and develop adequate management strategies of these diseases. For this purpose, 36 isolates of Fusarium were isolated from onion plants in 17 sites and subjected to molecular analysis and biometric characterization. The results revealed that the isolates belong to five Fusarium species: Fusarium oxysporum (44.44% of the isolates), Fusarium proliferatum (41.66%), Fusarium solani (5.55%), Fusarium fujikuroi (5.55%) and Fusarium thapsinum (2.77%). Fusarium oxysporum, F. proliferatum, F. solani and F. fujikuroi had faster mycelial development, with a growth rate of 7.72 - 8.27 mm/d, than F. thapsinum (6.52 mm/d). Conidia of F. oxysporum, F. proliferatum and F. solani were longer (4.74 - 5.96 μm) than those of F. fujikuroi and F. thapsinum (3.20 - 4.04 μm). Fusarium solani and F. oxysporum, respectively, had the largest and most partitioned conidia.

Genetic variation of sand flies(Diptera:Psychodidae)in Gampaha and Kurunegala districts of Sri Lanka:Complementing the morphological identification

Objective:To identity the variation of sand flies in the Gampaha and Kurunegala districts of Sri Lanka and to assess DNA barcoding as a complementing method for morphological identification.Methods:A total of 38441 sand flies were collected from selected localities in Gampaha and Kurunegala districts using standard entomological techniques from May 2017 to December 2018.Specimens were identified using morphological features and compared with mitochondrial cytochrome C oxidase subunitⅠgene-based DNA barcoding as an alternative tool.Results:Morphological and molecular identification confirmed the presence of four species under two genera(Phlebotomus and Sergentomyia).Phlebotomus argentipes was the predominant species,followed by Sergentomyia(S.)punjabensis,S.babu insularis,and an unidentified Sergentomyia sp.Phlebotomus argentipes showed a clear genetic differentiation from other species.S.babu insularis and S.punjabensis showed a higher genetic affinity to each other than the unidentified species.The unidentified Sergentomyia species is morphologically similar to S.zeylanica,but differs only in clavate gonostyle.Conclusions:DNA barcoding is an effective technique for the identification of sand flies.Further studies using molecular techniques will improve the knowledge of the cryptic diversity of Sri Lankan sand fly fauna.Establishing a reliable and standardized identification system for sand fly species in Sri Lanka is recommended.

甘肃省马铃薯枯萎病(Fusarium avenaceum)鉴定及其病原生物学特性

对甘肃省马铃薯枯萎病菌进行了分离、鉴定及培养特性的研究.结果表明,该病原菌大型分生孢子镰刀形,无色,多细胞,大小为（12.5～30）μm×（2.5～5）μm;小型分生孢子数量较多,无色,卵圆形或椭圆形,大小为（4.70～12.94）μm× （1.76～2.35）μm; ITS序列分析表明,病原菌与Fusarium avenaceum （JN6317483）亲缘关系最近,相似性达到100％,结合形态特征鉴定该病原菌为燕麦镰刀菌（Fusarium avenaceum）.该病原菌在5～40℃范围内均可生长,最适温度为20℃;供试的碳、氮源中,果糖和亮氨酸对该菌菌丝生长有显著的促进作用（P＜0.05）,而显著抑制菌丝生长的是氯醛糖和碳酸铵（P＜0.05）;不同类型培养基中,燕麦片培养基对菌丝生长有显著促进作用（P＜0.05）.

Antifungal Activity of Chitosan on Fusarium sulphureum in Relation to Dry Rot of Potato Tuber

The antifungal activity of chitosan on Fusarium sulphureum in relation to the inhibitory effect on dry rot of potato tuber was investigated. The results showed spore germination and mycelial growth of F. sulphureum were inhibited by chitosan treatment and the inhibitory effect was highly correlated with chitosan concentration used in this study. Morphological changes such as intertwisting hyphal, distortion, and swelling with excessive branching were observed by scanning electron microscopy （SEM） observation. Transmission electron microscopy （TEM） observation further indicated the ultrastructural alterations of hyphae. These changes included abnormal distribution of cytoplasma, non-membraneous inclusion bodies assembling in cytoplasm, considerable thickening of the hyphal cellular walls, and very frequent septation with malformed septa. Application of chitosan at higher concentration caused serious damage to fungal hyphae, including cellular membrane disorganisation, cell wall disruption, and breaking of inner cytoplast. New hyphae （daughter hyphae） inside the collapsed hyphal cells was often detected in the cytoplasm of chitosan-treated hyphae. In vivo tests showed that chitosan treatment at 0.5 or 1% effectively controlled the dry rot of potato tuber inoculated with a spore suspension ofF. sulphureum. However, the chitosan treatment at 1% caused phytotoxicity to potato tuber. This study suggests that the use of chitosan could be a promising handling as a natural fungicide to partially substitute for the synthetic fungicides in potato tuber.

冬虫夏草内生菌 Fusarium solani 化学成分研究

目的研究冬虫夏草内生菌Fusarium solani化学成分。方法Fusarium solani乙酸乙酯提取物采用硅胶、Sephadex LH-20、制备HPLC进行分离纯化,根据理化性质及波谱数据鉴定所得化合物的结构。结果从中分离鉴定出25个化合物。结论化合物1~17、19~24为首次从该真菌的次生代谢产物中发现。

海南省几种农作物枯萎病病原镰刀菌(Fusarium)初步鉴定

在分析国内外镰刀菌(Fusariumlink.)种类鉴定现状的基础上,运用培养性状和形态特征综合分析的方法,对海南省3个市县4种作物上采集的4个镰刀菌菌株进行了种类鉴定。结果鉴定出4个种,分别为:茄腐皮镰孢霉(Fusarium solani f.sp.),尖镰孢霉(Fusarium oxysporumf.sp.),萎蔫座镰孢霉(Fusarium bulbigenum f.sp.),稻恶苗镰孢霉(Fusarium moniliforme Sheldon)。

山西省重要作物萎蔫病病原镰刀菌(Fusarium)种类的鉴定(Ⅰ)

在分析国内外镰刀菌(Fusarium Link)种类鉴定现状的基础上,运用培养性状和形态特征综合分析的方法,对山西省三个地区七市县5种作物上采集的5个镰刀菌菌株进行了种类鉴定。鉴定出2个种,其中4个菌株属于镰刀菌属美丽组的尖孢镰刀菌(F.oxysporum Schl.),另一个菌株属于镰刀菌属李瑟组的串珠镰刀菌(F.moniliforme Shel-don)。5种作物病原镰刀菌种类分别是:棉花枯萎病菌(F.oxysporum)、西瓜枯萎病菌(F.oxysporum)、黄瓜枯萎病菌(F.oxysporum)、亚麻枯萎病菌(F.oxysporum)和玉米青枯病菌(F.moniliforme)。

Fusarium graminearum sensu stricto和F.asiaticum3种分子鉴定方法的比较

Fusarium graminearum sensu stricto和F.asiaticum是中国主要的禾谷镰刀菌复合种(FGSC)成员,为了提高这2个种的鉴定效率,选取48个引起小麦赤霉病和玉米茎腐病的FGSC菌株,比较了系统发育谱系分析、PCR-RFLP(聚合酶链式反应-限制性片段长度多态性)和多重PCR 3种分子鉴定方法。系统发育谱系分析首先扩增菌株的EF-1α和Tri101两个基因的部分序列,测序之后利用MEGA 6.06软件构建系统发育树,结果表明,有11个菌株与F.asiaticum聚在一起,37个菌株与F.graminearum sensu stricto聚为一枝,此法繁琐、复杂、成本高,但鉴定结果准确。PCR-RFLP基于组蛋白H3基因特殊的酶切位点,设计引物H3d StyⅠ/H3R1,通过PCR和酶切反应进行鉴定,电泳检测发现,有11个菌株为F.asiaticum,37个菌株为F.graminearum sensu stricto,该法工作量大且对操作过程要求高。多重PCR基于CYP51A基因设计特异引物Fa F/FaR和Fg F/FgR进行鉴定,电泳检测结果表明,有11个菌株为F.asiaticum,37个菌株为F.graminearum sensu stricto,此法操作简单、效率高。以上3种分子鉴定方法结果一致,但多重PCR方法具有快速、准确、高效的特点。

棉花枯萎病菌(Fusarium oxysporum f. sp. Vasinfectum)突变体的诱发与鉴定

通过对32个棉花枯萎病菌菌株进行的突变体诱发与鉴定实践及技术方法探索,进一步验证了可以采用KClO3培养基来诱发突变体,并利用各突变体对不同N源的利用能力差异而采用MM,MH,MA,MO2培养基对各突变体类型进行鉴定。实验结果显示绝大多数菌株(93.7%)在KClO3培养基上能诱发出突变体,但多数(85%)突变体类型不全,且不同菌株诱得突变体的难易不同,个别菌株很难诱发到突变体。各突变体的诱发率高低依次为nit1>nit3>nit8>nitM,与国内外相关报道结果基本一致。

In vitro Screening of Fusarium Wilt-resistant Germplasm Resources of Red Edible Seed Watermelon( Citrullus lanatus ssp. vulgaris var. megalaspermus Lin et Chao)

[ Objective] This study aimed to establish an appropriate technology system for in vitro screening of Fuzarium wilt-resistant germplasm resources of red edible seed watermelon and obtain variants resistant to fusaric acid, thus providing resistant materials for breeding Fusarium wilt-resistant red edible seed watermel- on. [ Method] Using Zhongxin No. 1 red edible seed watermelon advemitious buds as screening materials and fusaric acid （FA） as a stress agent, in vitro screen- ing of Fusarium wilt-resistant red edible seed watermelon clonal variants and identification of Fusarium wilt-resistance of the germplasm resources of red edible seed watermelon were performed. [ Result] The results showed that the appropriate FA for in vitro screening of Fusarium wilt-resistant red edible seed watermelon vari- ants was 15 mg/L. In vitro screening system for Fusarium wilt-resistant red edible seed watermelon variants was established preliminarily and FA-resistant regenera- ted plants were obtained. Among the 36 germplasm resources of red edible seed watermelon, there were 2 highly resistant materials, 6 moderately resistant materi- als, 11 slightly resistant materials and 17 highly susceptible materials. [ Conclusion] This study confirmed preliminarily that in vitro screening method is effective for obtaining resistant materials of red edible seed watermelon.

Wilt of Acer negundo L.caused by Fusarium nirenbergiae in China

Boxelder maple(Acer negundo L.)is widely grown as ornamental trees in China.In 2017 in Pizhou,Jiangsu Province,China,the boxelder maple trees suffered from a disease which caused wilting and yellowing leaves,rotted roots,and discolored xylem.The disease broke out from July to August.Seven fungal isolates were obtained from symptomatic tissue and tested for pathogenicity.Isolate An7 was isolated at high frequency from the discolored vascular tissues,which caused the wilt disease on healthy 2-year-old seedlings after inoculation and eventually killed all the seedlings.We conducted molecular analysis for An7,including the partial sequences of the rDNA internal transcribed spacer,calmodulin,RNA polymerase II second largest subunit and the translation elongation factor 1-αand evaluation of the variously sized conidia,chlamydospores and conidiophores and cultural traits.The pathogen was identified as Fusarium nirenbergiae,a member of the Fusarium oxysporum species complex.To our knowledge,this is the first report of F.nirenbergiae causing wilt on A.negundo in China.

Fumonisin-Producing Fusarium from Maize Grains in Tretep, Indonesia

Fusarium species commonly occur in maize are fungal pathogen which produce mycotoxins, such as fumonisin, trichothecene and zearalenone. In this study, Fusarium species were isolated from maize kernel from Tretep, maize producer region and were identified based on microscopic- and macroscopic characters as well as molecular characters using PCR assays and the partial sequence of TEF 1-α gene （Translation Elongation Factor 1-α. The fumonisin-producing ability of these Fusarium was determined by growing them in corn medium and analyzed their fumonisin by ELISA （enzyme-linked immuno assay）. Among 9 isolates, three of them were identified as Fusarium verticillioides, two as Fusarium temperatum, two as Fusarium globosum, one as Fusarium proliJeratum and one as Fusarium subglutinans. Fusarium temperatum is similar morphologically to F. subglutinans, however, both of their differences can be found by molecular analysis. Fumonisin-producing abilities of Fusarium were determined in concentrations 20.51 pg/g-1,109.74 pg/g medium with the highest producer was identified as F. globosum.

竹子内生真菌Fusarium oxysporum次级代谢产物的研究

[目的]对竹子内生真菌尖孢镰刀菌Fusarium oxysporum的次级代谢产物进行分离与鉴定。[方法]运用硅胶柱色谱、Sephadex LH-20凝胶色谱、制备液相色谱等方法进行分离纯化,根据理化性质和波谱技术对化合物的结构进行鉴定。[结果]从该菌的次级代谢产物中分离得到9个化合物,分别鉴定为1-(3, 4-二甲氧基苯基)-乙酮(1)、吲哚-3-乙酸乙酯(2)、吲哚-3-乙酸(3)、吲哚-3-乙酮(4)、吲哚-3-乙酸甲酯(5)、latifolicinin C(6)、白僵菌素(7)、1, 3-diamino-1, 3-dimethylurea(8)和2, 3-丁二醇(9)。[结论]除化合物3、7、9外,其余化合物均为首次从竹子内生真菌Fusarium oxysporum中分离得到。

北苍术枝枯病病原菌(Fusarium equiseti)的鉴定及其生物学特性研究

近年来,随着北苍术栽培面积的扩大,病害发生日趋严重。为了明确引起北苍术枝枯病病原菌及其生物学特性,利用柯赫氏法则、形态学特征及分子生物学技术对菌株进行了鉴定,并测定了菌丝生长最佳条件。结果表明,气生菌丝初期呈绒毛状至棉絮状、白色至粉红色、后期发展成驼色,分生孢子镰刀型、弯曲、中部细胞显著膨大、顶孢延长呈锥形、多数为3~7个分隔,厚垣孢子呈球形、成链状或单生于菌丝或孢子中。该致病菌的ITS序列与GenBank中木贼镰刀菌Fusarium equiseti(MK621018)的相似性为100%,结合形态特征与分子鉴定最终将其确定为木贼镰刀菌,GenBank登录号(MH290363)。该菌菌丝生长最适温度范围为25~30℃;菌丝在pH 4~11时均能正常生长,pH 8~11时生长最快;燕麦及玉米面培养基最适合该病原菌生长;光照对菌丝生长有很大影响,全光照条件菌丝生长最快;对碳源淀粉、葡萄糖及氮源酵母浸出粉利用率最高,培养7 d时菌落直径在7 cm左右。