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.

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.

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.

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.

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.

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。

Isolation and Identification of Anthracnose Pathogen from Fruit of Red Fuji Apple

The fruits of Red Fuji apple with anthracnose symptoms were collected and submitted to tissue isolation and culture. One strain of anthracnose pathogen （numbered as Acgl） was obtained, and it was identified by both morphological and molecular biological methods. According to the morphological characteristics of the colony and conidia and the results of rDNA-ITS sequence analysis, the Acgl strain was identified as Colletotrichum gloeosporioides.

Pathogen Identification of the Anthracnose of Ligusticum chuanxiong Hort. and Inhibitory Effect of Four Fungicides on the Pathogen

[Objective] The research aimed to isolate and identify the pathogen of anthracnose of Ligusticum chuanxiong Hort,and make the antifungal test on four kinds of fungicides for selecting the optimum fungicides.[Method] The pathogenic fungi was separated according to Koch＇s rule and the shape characteristics were identified. The antifungal test of fungicides was made by using plate mycelium growth inhibition method.[Result] The pathogenic fungi was separated from leaves of Ligusticum chuanxiong Hort. and morphological characters were consistent with that of Colletotrichum gloeosporioides. In addition to chloroisobromine cyanuric acid,the other three fungicides could inhibit the mycelium growth. The growth inhibition rates of 1 000 times Metalaxyl＋mancozeb,zineb,Thiophanate-methyl for the mycelium were 26.8%,22.1%,59.8% respectively after 7 days.[Conclusion] The anthracnose was caused by Colletotrichum gloeosporioides. Ligusticum chuanxiong Hort. was a new record host plant of Colletotrichum gloeosporioides and the best inhibiting fungicide was Thiophanate methyl.

辣椒炭疽病菌种类及主栽品种抗病性鉴定

辣椒炭疽病是辣椒生产上的重要病害,推广和使用抗病品种、采用合适的种植模式是辣椒生产上防治该病害最经济有效的方法。为明确河北省鸡泽县和望都县主栽品种辣椒炭疽病的病原菌种类和抗病性高低,采用稀释分离法分离鉴定了辣椒炭疽病的病原菌种类,采用田间自然发病方式测定了生产上10个主栽品种的抗病性程度,并调查两地不同种植模式辣椒炭疽病发病率。结果表明:鸡泽县和望都县辣椒炭疽病的主要病原菌是黑点炭疽菌(Colletotrichum capsici)和红色炭疽菌(Colletotrichum gloeosporioides)。10个辣椒品种对辣椒炭疽病没有高抗品种,望都椒和冀泽羊角红1号为抗病品种,病情指数分别为12.39和16.67。4种种植模式中以玉米与辣椒间作后辣椒炭疽病病果率最低,为8.89%。由此建议在选择抗病品种的基础上,通过辣椒与玉米间作预防辣椒炭疽病发生。

Bacterial extracts and bioformulates as a promising control of fruit body rot and root rot in avocado cv. Hass

At least 20-40% of annual losses of avocado crops are caused by pathogenic fungi.The chemical treatments of these diseases are inefficient,cause environmental pollution and are increasingly restricted by international laws.This work aimed to assess the biocontrol capacity of a bacterial extract to protect avocado fruits and plants from pathogen infections.Extracts from the bacterial isolate Serratia sp.ARP5.1 were obtained from liquid fermentations in a biorreactor.A body rot postharvest infection model with Colletotrichum gloeosporioides on fruits was developed.Moreover,packaging conditions were simulated using the bacterial extract and the commercial fungicide prochloraz as a positive control.Additionally,seedlings infections with Phytophthora cinnamomi were performed on two types of avocado(West Indian race and cv.Hass).The Area Under Disease Progress Curve(AUDPC) was recorded using the bacterial extract and a commercial product with fosetyl-aluminium as treatments.The bacterial extract significantly reduced infections by C.gloeosporioides on injured avocado fruits at 31.1 μg mL^-1.Intact fruits were also protected against body rot infections at the same concentration and showed no significant differences with the commercial fungicide.On the other hand,AUDPC in the seedlings was significantly reduced with the extract treatment at 3 μg mL^-1 compared to the control.However,a possible phytotoxicity effect of the extract was evidenced in the seedlings and confirmed by pathogen recovery and tests on Raphanus sativus seedlings.Finally,formulations of the extracts(emulsion and emulsifiable concentrate) were prepared,and bioactive stability was assessed for 8 wk.The emulsion formulates demonstrated very stable bioactivity against P.cinnamomi.The extract and the emulsion formulate showed promising results for the control of avocado pathogens.New bioproducts based on this type of active principles could be developed for the benefit of avocado industry.

Pathogen Isolation and Identification of a Serious Leaf Disease of Red Sandalwood (Pterocarpus santalinus) Seedlings

In the introduction and propagation of red sandalwood （Pterocarpus santalinus）, a serious leaf disease of its seedlings in winter and spring seasons was found, but the name of the disease and its pathogen species have not been reported. The pathogen isolated from infected leaves of 18-month-old seedlings was identi- fied as Colletotrichum gloeosporioides by morphological characteristics of colony and conidium, and analysis results of rDNA-intemal transcribed spacer sequence （ITS） of the strain. Pathogenicity test results further confirmed that C. gloeosporioides was the pathogen responsible for the infected leaves symptoms of red sandal- wood. However, the disease belongs to an atypical anthraenose. Control of the leaf diseases of red sandalwood seedlings was discussed.

Postharvest Biological Control of Apple Anthracnose by Bacillus subtilis BS80-6,A Mutant Strain through Ion Implantation

The original Bacillus subtilis was isolated from the soil of an apple orchard,and B. subtilis strain BS80-6,selected from Bacillus subtilis by ion implantation,was used as an antagonist for postharvest biological control against apple anthracnose（ colletotrichum gloeosporioides）. The mechanisms of action and efficacy of Bacillus subtilis strain BS80-6 against apple anthracnose caused by colletotrichum gloeosporioides were studied in vitro and on apples in controlled and semi-commercial conditions. An application of cell suspension（ 108 cells per mL） of the antagonist in artificial wounds of apples reduced growth of C. gloeosporioides after storage at different temperatures. The inhibitory actions of cell culture to hyphal growth and to spores germination were 86. 6% and 98. 65%,respectively,and the control efficacy of cell culture against the disease was 60. 34% at room temperature. The results showed that BS80-6 was more effective against apple anthracnose.The cell culture of BS80-6 received better control efficacy against apple anthracnose than culture filtrates and autoclaved cell. All treatments significantly inhibited the disease both in vitro and in vivo compared with control. Fruits treated with cell culture and stored at 10 ℃ had lower infection rate,more delayed formation of acervwlus and smaller lesion diameter than those stored at 20 ℃. There was better control efficacy in fruits inoculated with spores after application of BS80-6. The mechanisms of BS80-6 activity appeared to involve production of an antifungal substance,deformation of hyphal cell and disintegration of cell wall. Besides,BS80-6 could improve the activity of major defense enzymes of apple,such as peroxidase（ POD） and polyphenol oxidase（ PPO）.

胶孢炭疽菌微管末端结合蛋白CgEB1的功能初步分析

在橡胶树胶孢炭疽菌基因组中鉴定到一个编码微管末端结合蛋白的基因CgEB1。为了研究CgEB1在胶孢炭疽菌生长和对寄主致病力中的作用,本研究构建了该基因的敲除突变体菌株ΔCgEB1,并对其生长和致病等表型进行了进一步的分析。结果发现,CgEB1的缺失导致病原菌的营养生长、产孢及其对寄主橡胶树叶片的致病力都受到明显的抑制。通过对附着胞结构的观测,发现敲除突变体菌株ΔCgEB1中附着胞形成率和对洋葱表皮细胞的入侵率都显著下降。此外,与野生型菌株WT相比,敲除突变体菌株ΔCgEB1对盐胁迫更加敏感。以上结果表明CgEB1是胶孢炭疽菌重要的致病因子。

Colletotrichum species associated with cultivated citrus in China

There have been considerable advances in the understanding of species concepts in the genus Colletotrichum.This has lead to the need to carry out fresh surveys of Colletotrichum species associated with important hosts.Colletotrichum species are associated with Citrus plants as saprobes,important pre-harvest and post-harvest pathogens,as well as endophytes.In this study,a total of 312 Colletotrichum strains were isolated from leaves,shoots and fruits of cultivated Citrus and Fortunella species with or without disease symptoms across the main citrus production areas in China.The morphology of all strains were studied and multilocus(ACT,TUB2,CAL,GAPDH,GS,ITS)phylogeny established.Strains were from four important species complexes of Colletotrichum,namely C.gloeosporioides species complex,C.boninense species complex,C.acutatum species complex and a final group including C.truncatum,which was rare on Citrus species.The species belonging to the C.gloeosporioides species complex comprised C.gloeos porioides and C.fructicola,the C.boninense complex comprised C.karstii and a new species C.citricola and the C.acutatum complex included a new species,C.citri.The ability of strains to cause anthracnose on citrus fruits was tested by inoculation and strains of Colletotrichum gloeosporioides,C.fructicola and C.truncatum were pathogenic.

胶孢炭疽菌特有效应蛋白基因CgE23对产孢能力的影响

天然橡胶作为中国重要的工业原料和战略资源,其产量一直受到病害的影响,其中包括橡胶树炭疽病。胶孢炭疽菌(Colletotrichum gloeosporioides)和尖孢炭疽菌(Colletotrichum acutatum)作为橡胶树炭疽病的致病因子,均可以引起橡胶树炭疽病,但胶孢炭疽菌的致病性远强于尖孢炭疽菌的致病性。本研究通过比较胶孢炭疽菌和尖孢炭疽菌的基因组测序结果,利用RT-PCR技术扩增了一个胶孢炭疽菌特有效应蛋白基因,命名为CgE23。生物信息学分析表明,CgE23编码一条212个氨基酸的多肽,分子质量为22.7 kD,其中第1~第16位氨基酸是典型的信号肽序列,第106~第208位的氨基酸编码一个HNH保守结构域。依据同源重组和基因回补原理,构建了CgE23基因的敲除载体pCB1532-CgE23-Sur和互补载体Cgniad-CgE23-Flag-HPH,并通过PEG介导转化法将其转入到胶孢炭疽菌原生质体细胞,分别经过氯嘧磺隆(Sur)和潮霉素(HPH)抗性筛选和分子鉴定后,成功获得CgE23基因的敲除突变体ΔCgE23和基因互补突变株Res-ΔCgE23。进一步研究发现,ΔCgE23的孢子产量显著低于野生型和互补株Res-ΔCgE23,基因互补株Res-ΔCgE23的孢子产量与野生型并无明显差异。以上结果表明CgE23与胶孢炭疽菌的产孢能力有关。本研究结果可为进一步研究胶孢炭疽菌特有效应蛋白基因CgE23的功能和致病机制提供帮助。

胶孢炭疽菌CgFADBD基因敲除突变体的构建及其表型分析

胶孢炭疽菌引发的橡胶树炭疽病是橡胶树减产的重要原因之一。为了解析橡胶树胶孢炭疽菌的致病机理,在前期研究中对胶孢炭疽菌进行了基因组和转录组测序,预测了一个编码黄素腺嘌呤二核苷酸(FAD)结合蛋白的基因,命名为CgFADBD。本研究中,通过RT-PCR技术对CgFADBD基因全长编码区进行了克隆和生物信息学分析。结果表明,该基因编码一条由505个氨基酸组成的多肽,预测分子质量为54.39 kD。该蛋白含有典型的信号肽序列(第1~26位氨基酸)和一个保守的FAD结合域(第69~203位氨基酸),此外,不含任何跨膜结构域。根据融合PCR和同源重组原理,构建了CgFADBD基因敲除突变体ΔCgFADBD。菌落生长和孢子产量统计结果表明野生型菌株WT与ΔCgFADBD没有明显差异。致病力分析结果表明,ΔCgFADBD对橡胶树叶片的致病力明显小于WT,综合以上结果表明,CgFADBD与胶孢炭疽菌对橡胶树的致病力有关,与菌丝生长和孢子产量关系不大。本研究结果对解析胶孢炭疽菌对橡胶树的致病机制提供一定的参考。

What are the common anthracnose pathogens of tropical fruits?

t Species of Colletotrichum are associated with anthracnose of a wide range of host plants including cultivated and wild tropical fruits.The genetic and ecological diversity of species associated with wild fruits are poorly explored,as compared to those associated with pre and postharvest diseases of cultivated fruits.In the present study,isolates of Colletotrichum were obtained from commercially available cultivated fruits,wild fruits(from native trees in natural habitats)and a few herbaceous hosts collected in northern Thailand.These isolates were initially characterized based on analysis of complete sequences of nuclear ribosomal internal transcribed spacer(ITS),into the genetically defined species complexes of Colletotrichum gloeosporioides,C.acutatum,C.boninense and C.truncatum.The isolates were primarily identified in the C.gloeosporioides species complex,based on a strongly supported clade within the ITS gene tree and were further characterized using multi-gene phylogenetic analyses and morphology.Phylogenetic analyses of ITS,partial sequences of actin(ACT),calmodulin(CAL),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),glutamine synthetase(GS)andβtubulin(TUB2)genetic markers were performed individually and in combination.Colletotrichum gloeosporioides sensu stricto was identified from lime(Citrus aurantifolia)and rose apple(Syzygium samarangense).Colletotrichum fructicola was isolated from dragon fruit(Hylocerous undatus)and jujube(Ziziphus sp.).Colletotrichum endophytica was found only from an unknown wild fruit.We observed a considerable genetic and host diversity of species occurring on tropical fruits within the clade previously known as Colletotrichum siamense sensu lato.The clade consists of isolates identified as pre and postharvest pathogens on a wide range of fruits,including coffee(Coffea arabica),custard apple(Annona reticulata),Cerbera sp.,figs(Ficus racemosa)mango(Mangifera indica),neem(Azadirachta indica)and papaya(Carica papaya)and was the dominant group of species among most wild fruits studied.With the exception of one isolate from banana,which grouped in the C.siamense clade,all the other isolates were identified as Colletotrichum musae.A new species,Colletotrichum syzygicola,associated with Syzygium samarangense in Thailand,is introduced with descriptions and illustrations.This study highlights the need to re-assess the evolutionary relationships of Colletotrichum species occurring on cultivated and wild fruits with emphasis on their ecology and cryptic diversification including sampling at regional and global scales.