Quantitative detection and monitoring of Colletotrichum acutatum in strawberry leaves using real-time PCR

Real-time PCR assays for Colletotrichum acutatum , one of the most important pathogens of strawberry worldwide, were developed using primers designed to the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) and the β-tubulin 2 gene. Using TaqMan technology, the ITS-based assay could reliably detect as little as 50 fg genomic DNA, 100 copies of target DNA, or 25 conidia. The β-tubulin-based assay was c . 66 times less sensitive, and therefore less suitable for detection purposes. The TaqMan-ITS assay recognized all C. acutatum isolates tested from various intraspecific molecular groups, while no amplification was observed with several other Colletotrichum species or other strawberry pathogens, indicating the specificity of this assay. Detection and quantification of C. acutatum was demonstrated in artificially and naturally infected strawberry leaves. First, C. acutatum was detected in plant mixes of which only 0·001% of the tissue was infected by C. acutatum . Secondly, real-time PCR analysis of leaf samples taken at various times after inoculation indicated that the assay allowed monitoring of growth progression of C. acutatum . This real-time PCR-mediated monitoring of the pathogen was well-correlated with microscopic data, and confirmed that leaf age may play a role in the extent of C. acutatum infection. Finally, the assay allowed detection of C. acutatum in naturally infected and symptomless strawberry leaves collected from production fields and planting material.     

Sensitive real-time PCR detection of Xanthomonas fragariae in strawberry plants

Xanthomonas fragariae , the causal agent of angular leaf spot on strawberry, is a quarantine organism in strawberry propagation material in the European Union. For the reliable screening of planting material for latent infections, a real-time PCR assay based on Taqman® chemistry for the detection of X. fragariae was developed. Primers and probe sequences were based on a DNA fragment amplified by a previously reported X. fragariae -specific technique. The sequence of this genomic fragment had no significant similarity with any published GenBank sequence. Specificity of the designed assay was tested with an extended range of X. fragariae collection strains and isolates, with other Xanthomonas spp. and with unidentified bacterial isolates from strawberry plants. A nested PCR, which until now was the reference method for sensitive detection in planta , cross-reacted with the reference strain of Xanthomonas campestris pv. campestris . In combination with an elaborated DNA extraction procedure, the Taqman® PCR enabled reliable detection down to 300 colony forming units in a 100 mg strawberry leaf sample. The assay offers a new tool for epidemiological research and for sanitary control of plant material with low level or latent infections of X. fragariae .     

Management of strawberry anthracnose by choice of irrigation system,mulching material and host resistance

We studied, by means of field experiments, the combined effects of irrigation system, mulching material and genetic resistance on the incidence of strawberry anthracnose caused by Colletotrichum acutatum. All trials involved artificial inoculation with fruits colonized by the pathogen. One experiment examined the effects of drip irrigation, overhead sprinkler irrigation, and localized low-pressure tape irrigation, and the effects of grass (Brachiaria sp.) and plastic (polyethylene) mulches, in a split plot, randomized complete block design with four replicates. Lower disease incidence, indicating lower pathogen dispersal, was verified with low-pressure tape and drip irrigation. Flower blight and fruit rot incidence in drip-irrigated plots was minimal (ca. 2% of the disease levels in the plots otherwise irrigated). Grass mulch reduced flower blight in the sprinkler system (up to 80%), but had no significant effect when disease levels were already low due to use of other irrigation systems. A second experiment compared the effects of grass mulch, pine (Pinus elliotti) mulch, and plastic mulch with overhead sprinkler irrigation on strawberry cultivars partially resistant (‘Dover’) and susceptible (‘Campinas’) to the disease in a factorial completely randomized design (CRD). Both organic mulches reduced flower blight and diseased fruit incidence by up to 76% compared to the plastic mulch treatment, especially in the early stages of the epidemics. ‘Dover’ had 70% lower incidence of flower blight than ‘Campinas’, and the effects of organic mulches were more pronounced in ‘Campinas’. A third experiment, with all three irrigation systems combined with inoculated and non-inoculated plots, estimated yield effects due to disease and irrigation system. All plots were covered with plastic mulch in a CRD with six treatments. This experiment clearly confirmed the higher flower blight and fruit rot incidences when sprinkler irrigation was used. In inoculated treatments, fruit yield was significantly reduced in tape-irrigated plots, and more so in sprinkler-irrigated plots. In drip-irrigated plots yield was reduced by only 1%. Overall, our results confirm the importance of cultural and genetic factors as valuable means of strawberry anthracnose management. Adoption of localized irrigation systems, the use of organic mulches and choice of a partially resistant cultivar significantly reduced disease levels in field plots. The importance of water splash for C. acutatum dispersal and its dependency on the soil mulch characteristics are the likely causes for the results observed.     

实时荧光PCR检测瓜炭疽病菌

采用实时荧光PCR技术建立了瓜炭疽病菌(Colletotrichum orbiculare)的检测方法。根据瓜炭疽病菌甘油醛-3-磷酸脱氢酶(GAPDH)基因和谷氨酰胺合成酶(GS)基因序列,设计了该病菌特异性引物和TaqMan探针,并对所设计的引物和探针的反应条件进行了优化。采用本试验建立的实时荧光PCR方法对瓜上的其他菌株及近似菌株进行检测,可将瓜炭疽病菌与其他病原菌区分开。灵敏度试验表明,25μL体系中只要有39.6pg的核酸量就可以被检测到,检测灵敏度达到1.584pg/μL,比普通PCR检测灵敏度高100倍。同时对田间采集的病株和未知样品进行的检测证明了引物和TaqMan探针的特异性。     

Colletotrichum acutatum interactions with unripe and ripe strawberry fruits and differential responses at histological and transcriptional levels

Microscopic investigations were conducted into the interaction of Colletotrichum acutatum on white and red strawberry (Fragaria ×ananassa) fruit surfaces. The results showed that, whilst the early interaction events were similar in both white and red fruits, after 24 h fungal colonization dramatically varied: in white fruits C. acutatum became quiescent as melanized appressoria, but on red fruits it displayed subcuticular necrotrophic invasion. A microarray analysis of white and red strawberries after 24 h of interaction with C. acutatum was performed, in order to reveal differences in gene expression possibly related to the different susceptibility of unripe and ripe fruits. Epi/catechin‐related genes and fatty acid metabolism genes, involved in the production of quiescence‐related molecules such as flavan‐3‐ols, proanthocyanidins and antifungal dienes, were found to be regulated during strawberry ripening, supporting a role for these molecules as preformed defence mechanisms. Besides several genes commonly regulated upon pathogen interaction, different genes were specifically transcribed only in white or red challenged fruits; a number of these, such as those coding for lectin and polyphenol oxidase, possibly account for specific pathogen‐induced responses. The putative biological role of these genes in the different susceptibility of fruits to C. acutatum is discussed.     

Tracing Latent Infection of Colletotrichum acutatum on Strawberry by PCR

Colletotrichum acutatum, a quarantine organism on strawberries in the EU, was found in Finland for the first time in 2000. Concern about rapid, unnoticeable spread of this pathogen has necessitated studies to find methods with which the quiescent fungus infection can be detected in imported, cold-stored strawberry plant material. Successful detection of C. acutatum in strawberry tissues by polymerase chain reaction (PCR) is dependent on the method of DNA extraction used. Good-quality nucleic acid, free of PCR inhibitors, was successfully prepared by slightly modifying the DNA extraction method of a commercially available kit. Species-specific primers, previously described in the literature, were successfully used in the PCR reaction. C. acutatum was detected by PCR both on symptomatic and asymptomatic plant parts and in artificially and naturally infected strawberry tissues. Positive PCR results were obtained from ripe and unripe berries, runners, petioles and different parts of crowns. The data demonstrate that the PCR technique can be used to detect C. acutatum in strawberry tissue even in plant parts that do not show visible symptoms.     

UV-C照射对草莓炭疽病3种致病菌生活力和致病力的影响

炭疽病是草莓种植中普遍发生、危害严重的病害之一,而UV-C对真菌的生长繁殖有极大的影响。为了探究UV-C辐照对引起草莓炭疽病的3种主要病原菌生长和致病力影响的差异,以果生炭疽菌Colletotrichum fructicola、胶孢炭疽菌C.gloeosporioides和暹罗炭疽菌C.siamense为供试菌,分析UV-C辐照对其分生孢子存活、菌丝生长和产孢能力及对草莓叶片致病力的影响。结果显示,在105～420 J/m~2辐照剂量(辐照30～120 s)范围内,3种炭疽菌孢子的相对存活率对UV-C辐照剂量的响应存在显著差异,C.gloeosporioides的耐受性最强,C.fructicola最为敏感,辐照剂量420 J/m~2(辐照120 s)下,3种炭疽菌孢子的相对存活率在4%或以下,接近分生孢子的致死剂量。840～1 260 J/m~2辐照剂量(辐照4～6 min)下,C.fructicola与C.gloeosporioides菌丝生长对UV-C辐照的耐受性相当;1 680～2 520 J/m~2辐照剂量(辐照8～12 min)下,3种炭疽菌对不同时间的UV-C辐照的耐受性...     

Effects of nitrogen, phosphorus, potassium and calcium nutrition on strawberry anthracnose

The effects of a range of concentrations of four nutrients – nitrogen, phosphorus, potassium and calcium – in fertilizer solutions on the severity of anthracnose on strawberry cv. Nyoho cultivated under a noncirculation hydroponics system were determined after inoculation with Colletotrichum gloeosporioides . Crop growth and tissue nitrogen, phosphorus, potassium and calcium contents of the entire above-ground parts of the plant were also investigated. Elevated nitrogen and potassium concentrations in the fertilizer solution increased disease severity in contrast to phosphorus and calcium. Treatment with either NH₄ or NO₃ nitrogen was not significantly different. The dry weight of the strawberry plants increased significantly with elevated concentrations of nitrogen ( R ² = 0·9078) and phosphorus ( R ² = 0·8842), but was not influenced by the elevated amounts of potassium ( R ² = 0·8587) and calcium ( R ² = 0·6526) concentrations.     

Epidemiology,histopathology and aetiology of olive anthracnose caused by Colletotrichum acutatum and C. gloeosporioides in Portugal

Anthracnose is an important disease affecting mature olive fruits, causing significant yield losses, and poor fruit and oil quality. In Portugal, high anthracnose incidence was recorded during 2003–2007 with 41% of 908 orchards surveyed displaying disease symptoms. In another 14% of the orchards, the pathogen was recorded in symptomless plants. Disease severity was on average 36%, frequently reaching 100%. In Portugal, anthracnose is endemic to neglected orchards of susceptible cultivars, but under favourable conditions it can also severely affect less susceptible cultivars. Pathogens were genetically heterogeneous, with Colletotrichum acutatum genetic group A2 as the most frequent (80%), followed by group A4 (12%) and group A5 along with C. gloeosporioides (3–4%), while groups A3 and A6 of C. acutatum were sporadic. Important geographic variations were observed in the frequencies of these populations, accompanied by year‐to‐year populational shifts. Epidemiology and histopathology studies showed the presence of the pathogens on vegetative organs year‐round, particularly on olive leaves and branches, and on weeds. These represent inoculum reservoirs where secondary conidiation occurs, and conidia are then dispersed by spring rains reaching flowers and young fruits or by autumn rains reaching pre‐mature fruits. Unripe fruits were colonized without showing symptoms up to penetration of the cuticle, but further colonization and symptom production was completed only as fruits matured. These findings challenge current control practices, particularly the timing of fungicide treatment, and contribute to improved disease management.     

Two subpopulations of Colletotrichum acutatum are responsible for anthracnose in strawberry and leatherleaf fern in Costa Rica

Strawberry, Fragaria × ananassa, and leatherleaf fern, Rumohra adiantiformis, are two important crops in Costa Rica. One of the most severe diseases affecting these crops is anthracnose, caused by members of the fungal genus, Colletotrichum (teleomorph; Glomerella). Eighty single-spore isolates from strawberry and leatherleaf fern were identified as Colletotrichum acutatum by species-specific PCR, and were further characterised by Universally Primed PCR (UP-PCR) fingerprinting analysis, and sequence analysis of the ribosomal internal transcribed spacer (ITS) region. Morphological differences, genotypic variation revealed by UP-PCR fingerprinting analysis, and a single sequence polymorphism within the ITS2 region were found between the isolates from strawberry and leatherleaf fern, respectively. The UPGMA cluster analysis of the fingerprints clearly separated the isolates derived from strawberry and leatherleaf fern into two different clusters. Pathogenicity assays on detached strawberry fruits confirmed the apparent difference between the two groups of isolates. It is therefore suggested that the pathogens responsible for strawberry anthracnose fruit rot and leatherleaf fern anthracnose in Costa Rica, belong to two distinct subpopulations of C. acutatum.     

Characterization and epidemiology of Colletotrichum acutatum sensu lato (C. chrysanthemi) causing Carthamus tinctorius anthracnose

In 2012, Colletotrichum isolates were collected from field‐grown safflower (Carthamus tinctorius) crops in central Italy from plants exhibiting typical anthracnose symptoms. Colletotrichum isolates were also collected from seed surfaces and from within seeds. The genetic variability of these isolates was assessed by a multilocus sequencing approach and compared with those from Colletotrichum chrysanthemi and Colletotrichum carthami isolates from different geographic areas and other Colletotrichum acutatum sensu lato‐related isolates. Phylogenetic analysis revealed that all of the strains isolated from C. tinctorius belonged to the species described as C. chrysanthemi, whereas all of the strains belonging to C. carthami had been isolated from Calendula officinalis. Phenotypic characterization of isolates was performed by assessing growth rates at different temperatures, morphology of colonies on potato dextrose agar (PDA) and the size of conidia. All C. chrysanthemi isolates from safflower had similar growth rates at different temperatures, comparable colony morphologies when grown on PDA and conidial sizes consistent with previously described C. chrysanthemi isolates. Pathogenicity tests were performed by artificially inoculating both seeds and plants and confirmed the seedborne nature of this pathogen. When inoculated on plants, C. chrysanthemi caused the typical symptoms of anthracnose on leaves. This is the first record of this pathogen on C. tinctorius in Italy, and it presents an updated characterization of Colletotrichum isolates pathogenic to safflowers in Europe.     

梨火疫细菌实时荧光PCR和诱捕PCR-ELISA检测方法的建立

根据梨火疫细菌中独特而保守存在的质粒pEA29，设计了1对引物和3条探针，建立了实时荧光PCR检测方法和诱捕PCR-ELISA检测方法。实时荧光PCR采用带荧光标记的核酸杂交探针，边扩增边检测，步骤简单，不需PCR后处理，可避免假阳性和交叉污染；诱捕PCR-ELISA检测方法只需简单处理的样品就能检测，减少了核酸不纯出现的漏检，由于增加了核酸杂交探针，可不需凝胶电泳EB染色检测，不会出现假阳性问题。     

Latent entry and spread of Colletotrichum acutatum (species complex) in strawberry fields

Anthracnose, caused by Colletotrichum acutatum (species complex), has become a troublesome problem in strawberry production worldwide. This paper reports (i) an optimized sampling method combined with a real‐time PCR technique to detect the latent presence of C. acutatum in cold‐stored strawberry plants used as planting material in several European countries, and (ii) a study of the spread of C. acutatum following a point inoculation under field conditions. Screening of different parts of planting material suggested that C. acutatum is most likely to be present on runners and old petioles. In addition, in seven out of nine batches of planting material from different nurseries, latent infection by C. acutatum was detected in at least one of five replicate samples. Field experiments in 2009 and 2010 showed extensive latent within‐field spread of the pathogen on strawberry leaves, with a within‐row dispersal distance up to at least 1·75 m in 1 week. A straw ground cover between the rows did not decrease C. acutatum spread, probably because introduced (and/or subsequent) inoculum was confined to the plant bed (within the row) and was not present between the beds. Moreover, the number of C. acutatum spores on the symptomless leaves, as estimated using a real‐time PCR method, was significantly (P < 0·05) correlated with the incidence of fruit rot at harvest and post‐harvest (r = 0·56–0·66). These results illustrate the importance of detecting latent infections in planting material and strawberry leaves in the field.     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

Isolation and pathogenicity of Colletotrichum spp. causing anthracnose of strawberry in south west Spain

Anthracnose, caused by Colletotrichum spp., is a major disease of cultivated strawberry, Fragaria × ananassa. This study identifies the Colletotrichum spp. which causes strawberry anthracnose in the southwest of Spain. A survey of the region was carried out, and the strains isolated were identified as C. acutatum by using the polymerase chain reaction (PCR) with genus and species-specific primers, demonstrating that this species is currently the causal agent of strawberry anthracnose in the studied region. The pathogenicity of C. acutatum and C. gloeosporioides strains was evaluated on two principal strawberry cultivars (cvs Camarosa and Ventana) under field conditions, the latter being more pathogenic than the former.     

Components of genetic variation for resistance of strawberry to Phytophthora cactorum estimated using segregating seedling populations and their parent genotypes

Strawberry ( Fragaria × ananassa ) seedlings from 50 bi-parental crosses among 20 elite genotypes were evaluated for resistance to Phytophthora cactorum after artificial inoculation. Plots of seedlings or runner plants were rated using a disease severity score and the percentage of stunted plants per plot. The distribution of cross means for percentages of plants with stunting was highly skewed; 79% of the inoculated seedlings showed some level of stunting compared to non-inoculated control seedlings, and all but one of the crosses had 50% or more stunted plants. Disease severity scores for the bi-parental crosses were normally distributed and expressed a range of variation not reflected by the percentage of visibly stunted plants. Factorial analysis based on seedling plot means demonstrated significant additive genetic variance for the disease severity score, and the additive genetic variance was 1·9 times greater than the estimated dominance variance. The cross-mean heritability was     for the severity score. Estimates of the additive genetic variance component using the covariance of severity scores obtained from the seedling analysis and with severity scores for their parents evaluated in a commercial environment were similar,     and 0·30, respectively. Most of the selection response obtained through genotypic selection would thus be transferred to segregating offspring.     

Development of conventional and real-time PCR protocols for specific and sensitive detection of Colletotrichum capsici in chilli (Capsicum annuum L.)

Anthracnose, caused by Colletotrichum capsici, is a major disease of chilli (Capsicum annuum L.) affecting both fruit and seed quality. The pathogen is both internally and externally seedborne. However, a rapid and sensitive method for detection of this pathogen in seeds is currently limited. In this study, a polymerase chain reaction (PCR) method based on sequence characterized amplified region (SCAR) marker was developed for specific and sensitive detection of C. capsici in chilli seeds and fruits. The developed SCAR primers were highly specific to C. capsici and resulted in the amplification of an expected 250-bp fragment from genomic DNA of all seven of the C. capsici isolates tested. No amplification occurred when the SCAR primers were tested with genomic DNA from three other fungal isolates and four other Colletotrichum species. The SCAR primers successfully amplified similar sized fragments from DNA derived from C. capsici-infected chilli fruits. The molecular detection sensitivity of C. capsici was 1 pg of purified C. capsici DNA template and 25 ng of DNA from C. capsici-infected chilli fruits. A real-time PCR assay was also developed using SYBR Green chemistry for detection of C. capsici in chilli fruits and seeds. The standard curve obtained showed a linear correlation between copy number of the cloned target DNA sequence of C. capsici and cycle threshold (Ct) values, with R² of 0.98. These PCR-based assays may be highly useful in detection of this important pathogen in chilli seeds and fruits in plant quarantine laboratories.     

穿刺巴斯德芽菌荧光定量PCR检测方法的建立与应用

以穿刺巴斯德芽菌16S rDNA片段为靶标,通过对荧光定量PCR反应条件的摸索,建立该菌的荧光定量PCR检测方法.所选靶点最适退火温度60℃,正、反向引物的最佳浓度搭配为900、300 nmol/L;以Ct值和质粒拷贝浓度对数为坐标轴建立标准曲线,回归方程为y=-3.200×logx+ 34.43,R2值为0.998,PCR扩增效率为105.4％,对含穿刺巴斯德芽菌芽胞的土壤样品检测阈值为2×103个/g土壤.该方法敏感度高、特异性好,能够运用于穿刺巴斯德芽菌的定性、定量检测.     

香蕉穿孔线虫双重PCR快速检测技术研究

 香蕉穿孔线虫(Radopholus similis)是国际上公认的植物检疫性有害生物之-, 是热带地区果树上最重要的线虫。通过设计的属水平上的特异性引物R1、R2(R1/2)和种水平上的特异性引物RS1、RS2(RS1/2)进行双重PCR扩增香蕉穿孔线虫, 可扩增出长度为362和291 bp的2个特异性片段谱带。同时分别对检测体系中反应参数进行优化, 最佳延伸温度为51.4℃, 引物R1/2与RS1/2最佳浓度配比为0.2μmol·L^-1/1.2μmol·L^-1, 并测试检测引物的灵敏度, 能进行有效检测的最低起始核酸量为100 pg;同时, 也能对单条线虫进行检测以及样品中线虫的检测。专化性测试证明, 2对引物能有效地进行香蕉穿孔线虫的快速分子诊断。