Isolation and Identification of Colletotrichum gloeosporioides in Pears and Its Biological Characteristics

[Objective] This study aimed to explore the biological characteristics of Col etotrichum gloeosporioides in pears. [Method] Twenty-five C. gloeosporioides strains were isolated and identified from the diseased samples. Their pathogenicity was identified by inoculating the surface of punctured pears with fungal discs. The effects of different temperatures, pH values, carbon sources and nitrogen sources on the growth of C. gloeosporioides mycelia were explored by incubating fungal discs on the center of plates. [Result] Among the twenty-five C. gloeosporioides strains, three had strong pathogenicity, and eighteen had intermediate pathogenicity, and four strains had weak pathogenicity. Those highly-pathogenic strains had darker colonies, with dense mycelia, whereas those lowly-pathogenic ones had white colonies, with sparse mycelia. Those with fast-growing colonies showed strong pathogenicity, while those with slowly-growing colonies displayed weak pathogenicity. There was no relationship between conidia yield and pathogenicity. The optimum temperature for the growth of C. gloeosporioides mycelia was 25-30 ℃, and the optimum pH was 5.0-7.0. C. gloeosporioides could make use of various carbon sources （monosaccharide and disaccharide）, inorganic and organic nitrogen sources, and the optimal carbon source and nitrogen source were sucrose and beef extract, respectively. [Conclusion] Our study benefits further understanding of C. gloeospori-oides and helps to control pear anthracnose more effectively.

茶树炭疽菌 Colletotrichum gloeosporioides和C.acutatum的生物学特性比较及致病毒性初探

【目的】从重庆地区茶树种植区内的感病茶树叶片上分离纯化到2种能侵染茶树的炭疽菌,经形态学和多基因序列分析鉴定为C.acutatum和C.gloeosporioides,其中C.acutatum为本实验室从茶树上分离保存的新记录种。【方法】比较该2种炭疽菌的一般生物学特性。【结果】C.gloeosporioides的生长速度明显快于C.acutatum,但2种菌均能在多种培养基上进行生长,且同是以PDA和PSA为最适生长培养基,而菌生长的最适宜温度均在25~28℃之间,在40℃时仍能进行生长,菌丝致死温度在60~65℃之间。致病毒素研究发现2种茶树炭疽菌均能产生有致病力的外毒素,引起类似于自然状态下病原菌侵染形成的叶片坏死、萎焉等症状,但相比之下C.gloeosporioides的毒素活性更强。【结论】研究结果为后续茶树炭疽菌的致病性差异及其致病机制研究奠定了基础,也为炭疽菌的有效防治开辟了新的路径。

Carbendazim sensitivity in populations of Colletotrichum gloeosporioides complex infecting strawberry and yams in Hubei Province of China

The ascomycete fungus Colletotrichum gloeosporioides is a devastating plant pathogen with a wide host range and worldwide distribution. Carbendazim has been widely used to control anthracnose caused by the C. gloeosporioides complex in China for more than 30 years and resistance to carbendazim has been reported in China. A total of 125 Colletotrichum isolates of strawberry and yam were collected from different geographical regions in Hubei Province, China. Approximately 52.8％ of Colletotrichum spp. isolates showed resistance to carbendazim. The isolates tested in this study belong to four species, and the frequencies of resistant isolates differed across Colletotrichum species. Resistant isolates were found in C. siamense and C. fructicola. In contrast, all isolates of C. gloeosporioides and C. aenigma were sensitive to carbendazim. Highly carbendazim-resistant isolates harbored the E198A mutation in the β-tubulin 2 （TUB2） gene, whereas moderately carbendazim-resistant isolates harbored the F200Y mutation in the TUB2 gene. Carbendazim-sensitive Colletotrichum isolates in this study were not genetically similar enough to form a separate cluster from resistant isolates. The result of this study emphasizes the importance of knowing which Colletotrichum sp. is present, when strategies for disease control are made.

Generation and Analysis of Pathogenicity-related Gene Mutants of Colletotrichum gloeosporioides Using a Novel Promoter Trapping System

Agrobactedum tumefac/ens-mediated transformation （ATMT） is an efficient tool for insertional mutagenesis and is used in a wide variety of plants. This paper reports a promoter trapping method to generate mutants in the filamentous fungus, Colletotrichum gloeosporioides, by ATMT insertion of a trapping vector （pCAHPH） that carries a promoterless hygromycin phosphotransferase （hph） gone. Transformants were selected on the media containing 200 ~mL hy^omycin B, and screened for pathogenicity-related gene mdtants. Their pathogenicity-related mutants T-DNA flanking sequences were then cloned and analyzed. Hph genes were amplified from mutant genomic DNA but not from wild-type DNA, indicating that the phenotypic alternations of these mutants were the results of T-DNA inser- tion. T-DNA flanking sequences were obtained using modified themud asymmetric interlaced PCR. Two right-sided flanking sequences were highly homologous to proteins from other species.

Resistance risk and molecular mechanism associated with resistance to picoxystrobin in Colletotrichum truncatum and Colletotrichum gloeosporioides

Anthracnose,caused by Colletotrichum truncatum and C.gloeosporioides,is amongst the most serious diseases of soybean in China.Picoxystrobin,a quinone outside inhibitor fungicide,is commonly used for the control of anthracnose.Its resistance risk and mechanism in C.truncatum and C.gloeosporioides are unclear.In this study,the sensitivities of 128 C.truncatum and 121 C.gloeosporioides isolates to picoxystrobin were investigated,and unimodal distributions were observed with average EC_(50)values of 0.7740 and 1.1561μg mL^(-1),respectively.Eleven picoxystrobin-resistant mutants of C.truncatum and six mutants of C.gloeosporioides were acquired,with EC_(50)values varying from 5.40-152.96 and 13.53-28.30μg mL^(-1),respectively.Compared to the parental isolates,mutants showed similar or higher relative fitness in conidial production and germination,and pathogenicity.Collectively,the resistance risk of C.truncatum and C.gloeosporioides to picoxystrobin is moderate to high.There was positive cross-resistance between picoxystrobin and pyraclostrobin,but not between picoxystrobin and fluazinam,difenoconazole,or propiconazole.The G143S mutation in Cyt b protein was detected in seven high-resistant mutants of C.truncatum(RF>100),and G137R occurred in four moderate-resistant mutants(RF<_(50)).Contrastingly,there were no point mutations in Cyt b of any C.gloeosporioides mutants.Molecular docking confirmed that two mutations conferred different resistance levels to picoxystrobin.Under greenhouse trials,picoxystrobin did not control mutants with the G143S mutation,those bearing G137R or no point mutation were somewhat controlled,but at a lower level compared to wild-type isolates.These results showed that integrated management strategies should be implemented to preserve fungicide effectiveness.

Induced defense response in red mango fruit against Colletotrichum gloeosporioides

Mango fruit exposed to sunlight develops red skin and are more resistant to biotic and abiotic stresses.Here we show that harvested red mango fruit that was exposed to sunlight at the orchard is more resistant than green fruit to Colletotrichum gloeosporioides.LCMS analysis showed high amounts of antifungal compounds,as glycosylated flavonols,glycosylated anthocyanins,and mangiferin in red vs.green mango skin,correlated with higher antioxidant and lower ROS.However,also the green side of red mango fruit that has low levels of flavonoids was resistant,indicated induced resistance.Transcriptomes of red and green fruit inoculated on their red and green sides with C.gloeosporioides were analyzed.Overall,in red fruit skin,2,187 genes were upregulated in response to C.gloeosporioides.On the green side of red mango,upregulation of 22 transcription factors and 33 signaling-related transcripts indicated induced resistance.The RNA-Seq analysis suggests that resistance of the whole red fruit involved upregulation of ethylene,brassinosteroid,and phenylpropanoid pathways.To conclude,red fruit resistance to fungal pathogen was related to both flavonoid toxicity and primed resistance of fruit that was exposed to light at the orchard.

Antimicrobial activity of kojic acid from endophytic fungus Colletotrichum gloeosporioides isolated from Sonneratia apetala,a mangrove plant of the Sundarbans

Objective: To isolate and evaluate the antimicrobial activity of the active principle(s) from the ethyl acetate(EtOAc) extract of endophytic fungus Colietotrichum gloeosporioides(C.gloeosporioides) isolated from Sonneratia apetala. Methods: Water agar technique was used to isolate the fungus, and both microscopic and molecular techniques were used for identification of the strain. Potato dextrose broth was used to grow the fungus in large-scale. Reversed-phase preparative HPLC analysis was performed to isolate the major active compound, kojic acid. The EtOAc extract and kojic acid were screened for their antimicrobial activity against two Grampositive and two Gram-negative bacteria as well as a fungal strain using the resazurin 96-well microtitre plate antimicrobial assay. Results: The fungus C. gloeosporioides was isolated from the leaves of Sonneratia apetala. Initial identification of the fugal isolate was carried out using spore characteristics observed under the microscope. Subsequently, the ITS1-5.8 S-ITS2 sequencing was employed for species-level identification of the fungus C. gloeosporioides. Five litres of liquid culture of the fungus produced approximately 610 mg of a mixture of secondary metabolites.Kojic acid(1) was isolated as the main secondary metabolite present in the fungal extract, and the structure was confirmed by 1 D, 2 D NMR and mass spectrometry. The EtOAc extract and compound 1 exhibited considerable antimicrobial activity against all tested microorganisms.Whilst the minimum inhibitory concentration(MIC) values from the EtOAc extract ranged between 2.4×10^(-4)mg/mL and 2.5 mg/mL, those of kojic acid(1) were between 0.125 mg/mL and1 mg/mL. The EtOAc extract and kojic acid(1) were most active against Pseudomonas aeruginosa(MIC = 2.4×10^(-4). mg/mL) and Micrococcus luteus(MIC = 0.125 mg/mL), respectively. Conclusions:The results revealed that the endophytic fungus C. gloeosporioides could be a good source of commercially important kojic acid, which exhibited antimicrobial properties.

The different interactions of Colletotrichum gloeosporioides with two strawberry varieties and the involvement of salicylic acid

The disease symptoms recognized as‘Anthracnose’are caused by Colletotrichum spp.and lead to large-scale strawberry(Fragaria×ananassa Duchesne)losses worldwide in terms of both quality and production.Little is known regarding the mechanisms underlying the genetic variations in the strawberry–Colletotrichum spp.interaction.In this work,Colletotrichum gloeosporioides(C.gloeosporioides)infection was characterized in two varieties exhibiting different susceptibilities,and the involvement of salicylic acid(SA)was examined.Light microscopic observation showed that C.gloeosporioides conidia germinated earlier and faster on the leaf surface of the susceptible cultivar compared with the less-susceptible cultivar.Several PR genes were differentially expressed,with higher-amplitude changes observed in the less-susceptible cultivar.The less-susceptible cultivar contained a higher level of basal SA,and the SA levels increased rapidly upon infection,followed by a sharp decrease before the necrotrophic phase.External SA pretreatment reduced susceptibility and elevated the internal SA levels in both varieties,which were sharply reduced in the susceptible cultivar upon inoculation.The less-susceptible cultivar also displayed a more sensitive and marked increase in the transcripts of NB-LRR genes to C.gloeosporioides,and SA pretreatment differentially induced transcript accumulation in the two varieties during infection.Furthermore,SA directly inhibited the germination of C.gloeosporioides conidia;NB-LRR transcript accumulation in response to SA pretreatment was both dose-and cultivar-dependent.The results demonstrate that the less-susceptible cultivar showed reduced conidia germination.The contribution of SA might involve microbial isolate-specific sensitivity to SA,cultivar/tissue-specific SA homeostasis and signaling,and the sensitivity of R genes and the related defense network to SA and pathogens.

Rapid detection of the E198A mutation of carbendazim-resistant isolates in Colletotrichum gloeosporioides by loop-mediated isothermal amplification

Apple bitter rot is a serious agricultural disease caused by Colletotrichum gloeosporioides.In recent years,carbendazim-resistant C.gloeosporioides strains bearing an E198A point mutation in theβ-tubulin gene(GAG to GCG)have emerged,threatening global apple production.As such,rapidly detecting the presence of this E198Amutation in C.gloeosporioides isolates is essential in order to monitor the spread of this pathogen and to prevent outbreaks of disease.Herein,we developed a simple loop-mediated isothermal amplification(LAMP)approach to detecting the E198A mutation in C.gloeosporioides isolates from‘Gala’apple samples.This optimized LAMP protocol was sufficient to establish the E198A genotype of a given isolate following a 60min incubation at 63℃ by using four specific primers.The results of this reaction could be interpreted visually based on a fluorescent yellow-green color change upon the addition of the SYBR Green I dye,and were additionally confirmed via gel electrophoresis.Importantly,this LAMP assay was capable of rapidly and reliably detecting apples that were infected with carbendazim-resistant isolates harboring this E198A mutation.In conclusion,this LAMP assay in this study can rapidly,specifically,and sensitively detect cases of apple bitter rot caused by C.gloeosporioides isolates harboring the E198A mutation.

Biological Control Potential of <i>Colletotrichum gloeosporioides</i>for Coffee Senna (<i>Cassia occidentalis</i>)

A fungal pathogen, Colletotrichum gloeosporioides was isolated from a greenhouse-grown seedling of coffee senna (Cassia occidentalis) and evaluated as a mycoherbicide for that weed. Host range tests revealed that coffee senna, wild senna (C. marilandica), and sicklepod (C. obtusifolia) were also affected by this pathogen, but 35 other crop and weed species, representing 8 botanical families were not affected. The fungus sporulated prolifically on solid and liquid media with maximum spore germination and growth occurring at 20°C - 30°C. Optimal environmental conditions included at least 12 h of free moisture (dew) at 20°C - 30°C. Spray mixtures containing approximately 1.0 × 105 or more conidia·ml–1 gave maximum control when coffee senna seedlings were sprayed until runoff occurred. Coffee senna seedlings that were in the cotyledon to first-leaf growth stage were most susceptible to this pathogen. Weed control efficacy studies under field conditions demonstrated that control of coffee senna was directly proportional to the inoculum concentration applied. Results of these tests suggest that this fungus has potential as a mycoherbicide to control coffee senna, a serious weed in the southeastern U.S.

Effects of Boron on Spore Germination and Integrity of Colletotrictum gloeosporioides( Penz) Saec

Litchi anthracnose caused by Colletotrictum gloeosporioides （Penz） Saec. is an extremely destructive and widely distributed disease, which results in poor market value. Borate, an essential plant micronutrient that helps plant growth and has been used extensively in industry and agriculture as a safe method for control of fungi, was effective in the form of potassium tetraborate for control of C. gloeosporioides （Penz）. In this study, boron strongly inhibited spore germina- tion, germ tube elongation, and mycelial spread of C. gloeosporioides （Penz） in the culture medium. Application of boron at 1% caused the appearance of abnor- mal spores （disrupted） in some cases. On the basis of propidium iodide fluorescent staining, the loss of membrane integrity in C. gloeosporioides （Penz） was ob- served after boron treatment. Furthermore, Boron led to the leakage of cellular constituents （soluble proteins and carbohydrates） from hyphae of C. gloeosporioides （Penz）. These data suggest that the mechanisms may be directly related with the disruption effect of boron on cell membrane of the fungal pathogen, resulting in the breakdown of cell membrane structure and loss of cytoplasmic materials from the hyphae.

Study of the Action Mode of Wickerhamomyces anomalus against Colletotrichum gloeosporioides

Colletotrichum gloeosporioides is the causal agent of anthracnose disease in fruits and vegetables, representing a global problem. The use of biocontrol agents has proved effective against fungal diseases in a wide variety of products. In this work, the antifungal activity of Wickerhamomyces anomalus against C. gloeosporioides isolated from contaminated avocados was evaluated. The antagonism and volatile compound inhibition were measured on Petri dishes. In the mixed cultures, the mycelia damage was observed by scanning electron microscope （SEM）. Chitinase and glucanase production by the antagonism was quantified by the reducing sugars method, and biofilm formation was evaluated with 1% crystal violet. The yeast W. anomalus could reduce the growth of C. gloeosporioides up to 65% by direct antagonism and 10% by volatile compounds. The antagonist did not allow the conidia germination and mycelia growth in any of the tested formulations. SEM showed mycelial damage caused by W. anomalus. The antagonist showed adhesion to the mycelium by a polysaccharide biofilm. The presence of mycelium stimulated the hydrolytic enzyme production with the maximal activity of 21.4 U/mg for chitinases at 24 h and 10 U/mg for glucanases at 60 h. These results showed that W. anomalus used together different mechanisms to express its antifungal activity against C. gloeosporioides. This study might be the first report for this phytopathogen isolated from avocado fruits, which could represent an opportunity to establish biocontrol of diseases for this agricultural product.

Optimization and production of antifungal hydrolysis enzymes by streptomyces aureofaciens against Colletotrichum gloeosporioides of mango

We isolated naturally occurring actinomycetes with an ability to produce metabolites having antifungal property against, Colletotrichum gloeosporioides, the causal agent of mango anthracnose. One promising strain was strong antifungal activity, was selected for further studies. Based on the physiological and biochemical characteristics, the bacterial strain was identical to Streptomyces aureofaciens. Culture filtrate collected from the exponential and stationary phases inhibited the growth of fungus tested, indicating that growth suppression was due to extracellular antifungal metabolites present in culture filtrate. Isolate highly produced extracellular chitinase and β-1,3-glucanase during the exponential and late exponential phases, respectively. In order to standardize the metabolite production some cultural conditions like different incubation time in hours, pH, carbon sources and concentrations and nitrogen source were determined. During fermentation, growth, pH and hydrolysis enzymes production were monitored .Treatment with bioactive components exhibited a significantly high protective activity against development of anthracnose disease on mango trees and increased fruit yield.

Antimicrobial and cytotoxic activities of endophytic fungus Colletotrichum gloeosporioides isolated from endemic tree Cinnamomum malabatrum

In a survey of endophytic fungi associated with endemic plant Cinnamomum malabatrum leaves harbored a bioactive endophytic isolate CMS 3 was identified as Colletotrichum gloeosporioides through morphological and phylogenetic analysis based on ITS-rDNA.The ethyl acetate extract of fermentation broth of Colletotrichum gloeosporioides CMS 3 displayed antimicrobial activity against gram positive and gram negative bacteria as well as the fungal pathogen,Candida albicans.The ethyl acetate crude extract showed in vitro cytotoxicity against the HeLa,MCF-7 and MG63 cancer cell lines with the IC50 values of 94.2μg/ml,84.3μg/ml and 162μg/ml respectively.Gas chromatography and Mass Spectrophotometry(GC-MS)analysis of crude extract confirmed that CMS 3 was a prolific producer of secondary metabolites,in which nearly 74%of the metabolites not listed in the NIST database.Major compounds were phenol 3,5-dimethoxy acetate(11.82%),4'-isopropylidene-bis-(2-cyclohexyl)phenol,N-Didehydrohexacarboxyl-2,4,5-trimethylpiperazine and 1,2,4-Triazolium ylide.These metabolites may be responsible for its antimicrobial and cytotoxic activities.

芒果胶孢炭疽菌(Colletotrichum gloeosporioides)CgFAE基因对孢子产量及菌丝生长的影响

炭疽病是芒果的重要病害之一,由胶孢炭疽菌引起。阿魏酸酯酶为细胞壁降解酶,在致病机理中起着越来越重要的作用。本研究克隆了芒果胶孢炭疽菌的一个阿魏酸酯酶基因FAE,命名为CgFAE,该基因编码一个含有539个氨基酸的蛋白,分子质量约为58.3 k D。为了进一步研究该基因的功能,根据同源重组原理,构建了CgFAE基因的敲除载体pCB1532-CgFAE,通过PEG介导的原生质体转化法将其转入到胶孢炭疽菌原生质体细胞中进行同源交换,经过对转化子进行抗性筛选和分子鉴定,获得CgFAE基因的敲除突变体ΔCgfae,并对其孢子产量和菌落生长情况进行分析,发现胶孢炭疽菌CgFAE基因的敲除突变体ΔCgfae的孢子产量低于野生型菌株,菌落生长慢于野生型菌株。以上结果表明,该基因与胶孢炭疽菌的产孢能力与菌丝生长有关。

解淀粉芽胞杆菌JNC2摇瓶发酵条件优化

以菌体量和发酵滤液抑制柱花草炭疽病菌(Colletotrichum gloeosporioides)活性为指标,通过单因素和正交试验方法对解淀粉芽孢杆菌(Bacillus amyloliquefaciens)JNC2菌株的最适发酵培养基成分及发酵条件进行研究。结果表明:JNC2菌株最佳培养基为木糖2%,蛋白胨0.4%,酵母粉0.8%,氯化铵0.6%,硫酸镁0.2%;最佳发酵条件为初始pH 6.0,250 mL三角瓶装液量为60 mL,接种量体积分数7%,发酵温度34℃,转速200 r·min^–1,发酵时间72 h,优化后发酵滤液的抑菌活性显著提高了32.61%。

金钗石斛炭疽病病原鉴定及杀菌剂毒力测定

近年贵州赤水市种植的金钗石斛(Dendrobium nobile Lindl)炭疽病(烟煤病)发生较多,该病害分布广、危害严重。对金钗石斛炭疽病菌的生物学特性、培养性状及形态等研究,初步确定病原菌为胶孢炭疽菌(Colletotrichum gloeosporioides),结合ITS序列分析,确定该致病菌为胶孢炭疽菌。杀菌剂室内抑菌试验结果表明多菌灵对其有较好的抑制作用。

侵染油茶的两种病原菌的鉴定及其生物学特性研究

为了掌握湖北孝感本地油茶(Camellia oleifera)主要病害发生规律,采用常规组织分离法分离病原菌,依据病原菌的形态特征,结合其r DNA-ITS序列分析,对油茶病原菌进行分离和分子鉴定,并对其生物学特性进行了研究。结果表明,鉴定两种病原菌分别为胶孢炭疽菌(Colletotrichum gloeosporioides)和链格孢(Alternaria alternata);炭疽病菌菌株在PDA培养基中生长最快,最适碳源和氮源分别为淀粉、硫酸铵,p H 6.0为宜,最适生长温度为25℃,致死温度为52℃;链格孢菌株最适培养基为PDA培养基,以淀粉为碳源、硝酸铵为氮源为宜,p H 8.0时菌丝生长最快,最适温度为25℃,致死温度为54℃。

十三种杀菌剂对草莓胶孢炭疽菌的室内毒力测定

在实验室条件下,采用菌丝生长速率法测定了13种杀菌剂对发生在湖北的草莓胶孢炭疽菌(Colletotrichum gloeosporioides)的生物活性。结果表明,供试杀菌剂对草莓炭疽病菌菌丝抑制作用存在明显差异,对草莓胶孢炭疽病病菌毒力最强的是好立克、丙环.咪鲜胺和施保功,其EC50值分别为0.002 2、0.005 6和0.009 3 mg/L,福星、施保克、百泰、世高、凯特、溴菌腈和凯润的EC50值均相对很低,这10种杀菌剂均可作为防治草莓胶孢炭疽病的首选杀菌剂;腈菌唑、翠贝和二氰蒽醌的抑制活性较差,EC50值分别为12.12、17.58和106.99 mg/L。

气候、林分和管理因素对油茶炭疽病发生的影响

以大别山区安徽省舒城县河棚镇油茶林为试验地,对当地油茶炭疽病(病原Colletotrichum gloeosporioides)的发病情况与气候、林分及林地管理之间的关系进行了研究。结果表明,油茶炭疽病在温度20℃以上、降雨量大时发生严重;油茶与茶树的混交林病情指数最高,为21.0;油茶与板栗的混交林病情指数最低,为9.5;管理水平Ⅳ级(不做任何管理)的病情指数最高,为18.2;管理水平Ⅰ级(施肥、除杂、垦复松土、修除病枝和病虫害防治)的病情指数最低,为6.8;增施钾肥明显促进油茶果实的生长,果实炭疽病发病率比对照(不做任何处理)降低1/2左右;冬季整枝修剪可使果实发病率降低2/3左右。研究结果可为油茶炭疽病科学防治提供理论依据。