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.

芒果炭疽病菌,Colletotrichum gloeosporioides的生物学特性

芒果炭疽病菌Ｃｏｌｅｔｏｔｒｉｃｈｕｍｇｌｏｅｏｓｐｏｒｉｏｉｄｅｓ的营养生长、孢子形成、孢子萌发研究表明，营养生长和孢子形成的温度范围均为７～３７℃，适温范围分别为２０～３１℃，２５～３１℃。可以生长的ｐＨ值范围很广，以ｐＨ５．０～８．０生长良好；产孢的适宜ｐＨ为３．５～４．５。红光，兰光抑制营养生长，但光照有利于产孢，以萤光的促进作用最强。分生孢子在１０～４０℃均可萌发，适温范围为２５～３１℃。附着胞形成的温度范围为１５～３５℃，最适为３０℃。在相对湿度１００％＋水膜的条件下分生孢子萌发良好。营养有利于分生孢子萌发和附着胞形成。分生孢子萌发的ｐＨ为２～１１，最适ｐＨ为３～７，附着胞形成的最适ｐＨ值为３～６。

蓝光诱导的胶孢炭疽菌(Colletotrichum gloeosporioides)类胡萝卜素积累

胶孢炭疽菌(Colletotrichumgloeosporioides)为一种丝状真菌,蓝光照射可诱导类胡萝卜素的积累。光镜下观察表明,蓝光可诱导胶孢炭疽菌菌丝积累色素颗粒,而黑暗和红光处理却无此现象。类胡萝卜素的积累受蓝光光照强度的影响。28℃且蓝光为6.5μmol.m-2.s-1时,类胡萝卜素积累量可随光照时间延长呈增长趋势,在第5天达到最高峰为71.8μg/g FW,随后含量下降。此外,胶孢炭疽菌在黑暗中预培养的时间也影响蓝光的诱导反应。

芒果炭疽病［Colletotrichum gloeosporioides（Penz）］生物学及其综合防治研究

在云南景谷芒果种植区，芒果炭疽病对芒果树嫩叶、嫩梢、花穗和果实为害率分别为８５．７％，６３．４％，７７．１％和７３．３％。风雨和昆虫媒介是传播的主要方式。发生为害最适温度为２２℃～２５℃，相对湿度为８５％～１００％。该病在景谷种植区全年均可发生为害，主要为害在发生期。发生前期为害，是花穗干瘪和幼果掉落的原因之一。采取综合防治措施，特别在发生前期适时防治，可控制发生为害。

臭椿炭疽病菌盘长孢刺盘孢(Colletotrichum gloeosporioides Penz.)生物学特性研究

本研究测定了不同培养基、碳源、氮源、温度、pH条件下臭椿盘长孢刺盘孢(Colletotrichum gloeosporioides Penz.)菌株SQD-107的生长、产孢和孢子萌发情况。该菌株在PSA培养基上生长最快、产孢最多;在供测碳源中病原菌对蔗糖、葡萄糖利用较好,供测氮源中对硝酸铵、氯化铵利用较好。菌丝生长的适宜温度范围为10～35℃,最适25℃;产生分生孢子的适宜温度范围为10～30℃,最适25～30℃;孢子萌发的适宜温度范围为5～35℃,最适25℃。在pH3～10的范围内该菌均能生长和产孢,菌丝生长最适pH7～8;产生分生孢子最适pH为7,孢子萌发最适pH6～8。孢子的致死温度为60℃,持续时间10min。

柑桔叶面炭疽病Colletotrichum gloeosporioides附着胞的研究

在自然条件及人工接种外观无症柑桔叶片表明,柑桔叶片表面存在大量炭疽病菌的附着胞。人工接种幼苗的叶片上虽有大量附着胞存在,但经23个月后仍未见显症发病现象。接种植株上的新叶未见病菌附着胞。叶片组织解剖未发现病菌菌丝,这说明柑桔炭疽病潜伏侵染结构是普遍存在于柑桔叶面的附着胞。

辣椒炭疽病菌Colletotrichum gloeosporioides对啶氧菌酯的敏感基线及抗性突变体生物学性状

为评估辣椒炭疽病菌Colletotrichum gloeosporioides对啶氧菌酯的抗性风险,建立了辣椒炭疽病菌对啶氧菌酯的敏感基线,室内筛选获得了辣椒炭疽病菌抗啶氧菌酯突变体,并对突变体生物学性状进行了研究。在山西省3个未使用过啶氧菌酯及QoI类药剂的地区采集并分离到45株辣椒炭疽病菌。采用菌丝生长速率法测定其对啶氧菌酯的敏感性,结果显示,EC_(50)值介于1.404~16.650μg/mL,平均EC_(50)值为(6.783±3.499)μg/mL。啶氧菌酯与水杨肟酸(SHAM)共同处理时(SHAM的处理浓度为100μg/mL),EC_(50+S)为0.022~0.275μg/mL,平均(0.109±0.058)μg/mL,EC_(50+S)呈连续性单峰曲线,且敏感性频率分布呈近似正态分布,EC_(50+S)平均值可作为辣椒炭疽病菌对啶氧菌酯的敏感基线。且水杨羟肟酸旁路氧化贡献值F(F=EC_(50)/EC_(50+S))最低为6.039,最高为301.441,平均78.026。室内诱导获得8株抗性突变体,其中低抗突变体6株,中抗突变体2株。中抗突变体无性繁殖10代,其抗性可稳定遗传。突变体产孢量和菌丝生长速率与敏感菌株相比均无显著差异,但无药条件下,突变体致病力较敏感菌株有所降低。抗感菌株均以淀粉作为碳源、硝酸钾作为氮源时利用率最高;最适pH均为5和6;菌丝的最适生长温度均为25℃。表明辣椒炭疽病菌抗啶氧菌酯突变体具有较高的适合度,对啶氧菌酯具有较高抗性风险,这一研究结论为指导生产用药,延缓抗药性发展提供理论依据。

柑桔炭疽病菌Colletotrichum gloeosporioides抗药力试验

常用杀菌剂对柑桔炭疽病菌分生孢子杀伤力高达100%,可以阻止它的萌发,但对叶面潜存的附着胞效果很差。附着胞只能在植株器官组织衰弱条件下才能萌发侵入。因此,改善栽培管理条件、增强树势、提高抗病力就能有效地控制炭疽病的发生。

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。

枸杞内生真菌对胶孢炭疽菌Colletotrichum gloeosporioides的拮抗作用及生防潜力

为了明确枸杞内生真菌对胶孢炭疽菌的抑制作用,通过室内抑菌试验和离体试验测定了内生真菌对胶孢炭疽菌的抑制作用及对枸杞炭疽病的防治效果。结果表明,镰刀菌属Fusarium菌株NQ8GII4、NQ8GII7、NQ7GII4和篮状菌属Talaromyces菌株NQ6GIII11对胶孢炭疽菌有明显的拮抗作用。平板对峙试验显示,上述4株枸杞内生真菌具有较强的营养和空间竞争能力,菌株NQ8GII4对病原菌的抑制率高达93.43%。显微观察发现,枸杞内生真菌能缠绕并穿透胶孢炭疽菌菌丝,使胶孢炭疽菌菌丝膨胀崩解。菌株NQ7GII4的培养滤液在浓度为15%时对病原菌菌丝的抑制率达到70.75%,菌株NQ8GII7产生的挥发性物质对胶孢炭疽菌抑制率高达83.44%。离体生防试验结果表明,用拮抗性内生真菌预先占位接种枸杞嫩果及叶片,能有效抑制枸杞炭疽病菌的侵入和病斑的扩展,菌株NQ8GII410%培养滤液对枸杞炭疽病的防治效果最强,防效达到了92.54%和95.57%,说明枸杞内生真菌具有很好的生防潜力和应用前景。

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.

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.

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.

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.

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.

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.

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.

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.