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 .     

Conventional PCR and Real-time Quantitative PCR Detection of Helminthosporium Solani in Soil and on Potato Tubers

Silver scurf is an economically important blemish disease of potato caused by the fungus Helminthosporium solani. Two sets of PCR primers, Hs1F1/Hs2R1 (outer) and Hs1NF1/Hs2NR1 (nested) were designed to unique sequences of the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions of H. solani. Nested PCR was used to increase the specificity and sensitivity of single round PCR. Each primer set amplified a single product of 447 bp and 371 bp respectively, with DNA from 71 European and North American isolates of H. solani, and the specificity of primers was confirmed by the absence of amplified product with DNA from other fungal and bacterial plant pathogens. A simple and rapid procedure for direct extraction of DNA from soils and potato tubers was modified and developed to yield DNA of a purity and quality suitable for PCR within 3 h. The sensitivity of PCR for the specific detection of H. solani in seeded soils was determined to be 1.5 spores g^–1 of soil. H. solani was also detected by PCR in naturally infested soil and from peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed using the original primer sequences to perform real-time quantitative (TaqMan) PCR. The same levels of sensitivity for specific detection of H. solani in soil and tubers were obtained during first round mboxTaqMan-based PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR assay allows an accurate estimation of tuber and soil contamination by H. solani, thus providing a tool to study the ecology of the organism and to serve as a crucial component for disease risk assessments.     

Identification of <Emphasis Type="Italic">Globodera rostochiensis</Emphasis> and <Emphasis Type="Italic">G. pallida</Emphasis> in the Ukraine by PCR

Multiplex polymerase chain reaction was used to identify the potato cyst nematodes in soil samples from the Ukraine. The results show the occurrence of Globodera pallida in the Uzhhorod region (Zakarpatska oblast), where only G. rostochiensis had been previously reported. In the mixed potato cyst nematode (PCN) populations, G. pallida was less prevalent (2–5%) than G. rostochiensis (95–98%). A phylogenetic analysis based on ribosomal DNA internal transcribed spacer sequences showed that the Ukrainian population of G. pallida had >99% sequence identity with other G. pallida pa2/3 isolates from Europe. This study has demonstrated that polymerase chain reaction-mediated amplification of specific regions of the potato cyst nematode genome is not only highly effective as a species diagnostic tool but is also a sensitive method which can be used for taxonomic purposes with cyst collections which vary in age.     

Comparison of conventional and molecular methods for the detection of Rosellinia necatrix in avocado orchards in southern Spain

White root rot, caused by Rosellinia necatrix , is one of the most important diseases in avocado orchards and is particularly widespread on the Mediterranean seaboard of southern Spain. In this study, the presence of the pathogen in soil samples collected from the base of 47 plants showing different symptoms of canopy decline was assessed with a molecular detection method based on real-time Scorpion PCR. Results were compared with symptoms in the canopy and with the traditional method of isolation of R. necatrix from roots and/or bark. The fungus was isolated from 24 samples by the traditional method and from 37 soil samples by the molecular method (cycle threshold values 25·8 to 47·1), demonstrating the higher sensitivity and reliability of the molecular method. A single real-time PCR amplification was sufficient to detect R. necatrix in naturally infested soils. The avoidance of nested PCR has important practical implications because of the reduced costs and risk of cross contamination. Also, it enables faster sample analysis and is more appropriate for quantitative detection. A modified molecular method was also developed to detect R. necatrix in roots and in soils with very low populations of the pathogen.     

Detection and identification of Phytophthora fragariae Hickman by the polymerase chain reaction

Phytophthora fragariae Hickman, which causes strawberry red stele and raspberry root rot, is a quarantine organism for which specific and sensitive detection methods are required to test the health of planting material. Sequences of the internal transcribed spacer regions of the ribosomal gene repeat (rDNA) were used to develop primers for P. fragariae in a nested Polymerase Chain Reaction (PCR). The fungus was readily detected in infected but symptomless roots by nested, but not single-round, PCR. It was also detected in infested water samples obtained from the Dutch General Inspection Service by nested PCR. Detection of PCR products was at least 10-fold more sensitive by PCR-ELISA than by conventional visualisation on agarose gels.     

香蕉黑腐病菌(Botryodiplodia theobromae)的PCR检测

 根据香蕉黑腐病菌可可球二孢菌(Botryodiplodia theobromae)与其它香蕉病原真菌核糖体基因转录间隔区(rDNA-ITS)ITS1和ITS2间序列差异,设计了特异引物Bth-S(5'-TCTCCCACCCTTTGTGAAC-3')和Bth-A(5'-AAAAGT-TCAGAAGGTTCGTC-3'),利用此引物对包括可可球二孢菌在内的21个菌株基因组DNA进行PCR扩增,结果只有4个可可球二孢菌菌株扩增到422bp特异带,其它17个菌株无扩增产物。灵敏度测试结果表明此特异引物能对1pg的可可球二孢菌基因组DNA进行扩增。对自然感染黑腐病的香蕉果实组织和接种可可球二孢菌或多种香蕉病原真菌混合接种的果实组织进行检测,Bth-S和Bth-A引物对不仅能够在自然感染黑腐病果实组织中特异检测到可可球二孢菌,而且能在未显症和发病的接菌香蕉果实组织中特异检测得到可可球二孢菌。这为香蕉可可球二孢菌潜伏侵染检测提供了技术支持。     

土壤中烟草根黑腐病菌的实时定量PCR检测技术研究

 Thielaviopsis basicola is a soil-borne plant pathogen which causes root rot disease in tobacco plants. Detection and monitoring of T. basicolain soil is of great significance to control this disease. Based on the differences in internal transcribed spacer (ITS) sequences of T. basicola and other fungal pathogens, a specific primer pair Tb1/Tb2 for T. basicolawas developed. The results showed that the primer pair gave a single amplicon of 330 bp from T. basicola and revealed no undesirable cross-reaction with other seven soil-borne pathogen isolates and three tobacco rhizosphere dominant fungi isolates. With a series of 10-fold genomic DNA dilutions of T. basicola, the detection limit of 1 pg/μL in conventional PCRand100 fg/μL in real-time quantitative PCR was achieved. With DNA from the soil inoculated with different numbers of T. basicola conidia, the detection limit was 10 conidia per reaction in conventional PCR and 0.4 conidia per reaction in real-time quantitative PCR.     

Inoculation of Lupinus luteus with White Root Rot Fungus, Rosellinia necatrix, to Estimate Virulence

An inoculation method using Lupinus luteus was developed for estimating virulence of isolates of the white root rot fungus, Rosellinia necatrix Prillieux. Fungal cultures grown on pieces of mulberry twigs were placed in contact with the hypocotyl of 3-week-old seedlings growing in pots of soil. Disease development was uniform and reproducible in repeated experiments. Of 24 isolates with double-stranded RNA, eight were weakly virulent. This method is useful throughout the year for estimating virulence of many isolates of the fungus and for screening for hypovirulent isolates. Received 2 March 2001/ Accepted in revised form 25 May 2001     

Detection and Quantification of Spongospora subterranea in Soil, Water and Plant Tissue Samples Using Real-Time PCR

A sensitive real-time polymerase chain reaction (PCR) assay was developed for the quantification of Spongospora subterranea, the cause of powdery scab and root galling in potato, and the vector of Potato mop top virus. A specific primer pair and a fluorogenic TaqMan® probe were designed to perform a quantitative assay for the detection of S. subterranea in soil, water and plant tissue samples. The assay was tested using DNA from cystosori, zoospores, plasmodia and zoosporangia of the pathogen. DNA was extracted directly from cystosori suspended in water and from clay soil with varying levels of added cystosori. DNA obtained from zoospores released into nutrient solution by cystosori in the presence of tomato bait plants was also tested, as was DNA from plasmodia and zoosporangia in infected tomato roots. In many cases, detection was successful even at low inoculum levels. This specific quantitative assay could therefore be a useful tool for studying the biology of S. subterranea, and for the optimisation of disease avoidance and control measures.     

Detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. palmivora</Emphasis> in citrus roots using PCR-RFLP in comparison with other methods

Phytophthora nicotianae and P. palmivora are the most important soil-borne pathogens of citrus in Florida. These two species were detected and identified in singly and doubly infected plants using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS) regions of ribosomal DNA. The sensitivity of the PCR-RFLP was analyzed and the usefulness of the method evaluated as an alternative or supplement to serological methods and recovery on semi-selective medium. In a semi-nested PCR with universal primers ITS4 and ITS6, the detection limit was 1 fg of fungal DNA, which made it 1000× more sensitive than a single-step PCR with primers ITS4 and DC6. The sensitivity of detection for P. nicotianae was shown to be ten-fold lower than for P. palmivora, limiting its detection with restriction profiles in plants infected by both fungal species. Phytophthora nicotianae was detected with species-specific primers in all samples inoculated with this species despite the absence of species-specific patterns in RFLP. In contrast, the incidence of detection of P. palmivora in the presence of P. nicotianae was considerably lower using plating and morphological detection methods. Due to its high sensitivity, PCR amplification of ribosomal ITS regions is a valuable tool for detecting and identifying Phytophthora spp. in citrus roots, provided a thorough knowledge of reaction conditions for the target species is established prior to the interpretation of data.     

新疆加工番茄腐霉根腐病病原鉴定及其rDNA的ITS区段分析

为了明确新疆加工番茄根腐病的病原种类,从成苗期到果实成熟期对各主栽区的病原物进行分离,并进行形态学鉴定和ITS序列分析.结果表明,从162个根腐病样中获得120个分离物,其中腐霉菌93株,占总分离物的77.5%.腐霉菌分离物鉴定为3个种,瓜果腐霉Pythium aphani-dermatum(Edson)Fitzp,56株,占腐霉分离物的60.22%;简囊腐霉P.monospermum Pringsheim,6株,占6.45%;棘腐霉P.acanthicum Drechsler,2株,占2.15%;另有29个分离物因未诱发出雄器和藏卵器而无法鉴定到种.     

Sequence Analysis and Detection of Ralstonia solanacearum by Multiplex PCR Amplification of 16S–23S Ribosomal Intergenic Spacer Region with Internal Positive Control

Polymerase chain reaction (PCR) methods for detection and differentiation of Ralstonia solanacearum strains were compared. The 16S–23S rRNA gene ITS sequence data revealed the two main sequence clusters (divisions I and II) of R. solanacearum and further subclusters of division II. Based on this sequence data, primers were designed which differentiated divisions I and II. Furthermore, to improve reliability of the PCR assay for routine detection of R. solanacearum in host plants, a novel multiplex PCR assay was developed in which the pathogen-specific sequences are coamplified with host plant DNA as an internal PCR control (IPC). The assay was validated during routine testing of potato samples submitted in official surveys. Of 4300 samples from 143 cultivars, 13 tested positive in both multiplex PCR and immunofluorescence (IF) assays and could be confirmed by bioassay in tomato seedlings and reisolation of the pathogen. The IPC was successfully amplified from all samples tested. A further 12 samples gave positive IF results which were not confirmed by either the multiplex PCR or tomato bioassay, indicating a greater specificity of the latter two assays.     

Pathogenicity of Phytophthora austrocedrae on Austrocedrus chilensis and its relation with mal del ciprés in Patagonia

Field observations, isolations and pathogenicity tests were performed on Austrocedrus chilensis (Cupressaceae) trees to determine the pathogenicity of Phytophthora austrocedrae and its role in the aetiology of the cypress disease mal del ciprés (MDC) in Argentina. It was found that P. austrocedrae is a primary pathogen of A. chilensis. It was isolated from large necrotic lesions in the inner bark, and superficially in the sapwood, at the root collar and stem, in most of the MDC‐affected stands surveyed along the range of A. chilensis in Argentina. The main symptom in naturally infected trees was a necrotic lesion extending from killed roots up to 1 m up the tree bole. Seedlings, saplings and adult trees were all susceptible to inoculation with P. austrocedrae. Under favourable experimental conditions (flooding), inoculated seedlings suffered massive mortality in less than a month. The importance of diseases caused by Phytophthora spp. in South American forests is discussed.     

Quantification of seedborne infection by Rhynchosporium secalis in barley using competitive PCR

The potential of the competitive polymerase chain reaction (PCR) assay for quantification of seedborne infection by Rhynchosporium secalis in barley was examined using a primer set (RS1 and RS3) derived from the internal transcribed spacer (ITS) regions of ribosomal RNA genes of this pathogen. Introduction of a heterologous internal control, which competes for the same primer set in the conventional PCR assay, allowed for detection and quantification of R. secalis fungal biomass. In order to generate a standard calibration curve, DNA prepared from infected seeds with different levels of R. secalis infection was subjected to competitive PCR assay. The resulting PCR product ratio for each PCR reaction ( R. secalis -amplified DNA/internal control template-amplified DNA) increased proportionally with increasing levels of infected seed DNA in the reaction mixture. Naturally infected seed lots collected from 1995 to 1999 were used to demonstrate the potential of the competitive PCR assay as an alternative seed health testing method. The results from this competitive PCR assay were compared with those from conventional visual disease assessment and an agar plate assay. Although relatively good correlation between visual disease assessment and the competitive PCR was found in the case of artificially mixed seed samples, there was poor correlation in the experiments using naturally infected seed samples.     

Development of Specific PCR Primers for Identification and Detection of Rhizopycnis vagum

Rhizopycnis vagum is a recently described coelomycete known to belong to the complex of root rot pathogens contributing to vine decline of cucurbits in several parts of the world. However, the fungus has also been reported to infect tomato, and as an endophytic associate of mycorrhizal roots of wild, asymptomatic Pinus halepensis and Rosmarinus officinalis plants in Italy. To accelerate epidemiological and ecological investigations on this fungus, a PCR primer pair was developed. Primers Rv1-F and Rv1-R were designed, based on alignment of internal transcribed spacer (ITS) sequences (ITS1-5.8S-ITS2), which amplified a 396-bp fragment from all R. vagum isolates tested, including isolates pathogenic to melons and endophytic isolates from mycorrhizae. Specificity of the primer pair was verified both in silico (BLAST searches using each primer string as a query) and in PCR assays, where the primers failed to amplify DNA from any isolate of fungi taxonomically related to R. vagum (e.g. Massarina walkeri and Stagonospora spp.) and other vine decline and common soilborne pathogens (e.g. Monasporascus cannonballus, Acremonium cucurbitacearum, Fusarium spp. and Rhizoctonia solani). Under optimum conditions, detectable amplification of the specific sequence required 0.05 pg of target DNA. Amplification of the expected 369-bp fragment was also obtained from DNA root extracts of nearly asymptomatic Cucumis melo plants inoculated with R. vagum under greenhouse conditions.     

Specific Behaviour of French Aphanomyces Euteiches Drechs. Populations For Virulence and Aggressiveness on Pea, Related to Isolates from Europe, America and New Zealand

The pathogenic variability of Aphanomyces euteiches on pea was investigated using a collection of 88 pea-infecting isolates from France and 21 isolates from Denmark, Sweden, Norway, USA, Canada and New Zealand. Aggressiveness and virulence were assessed by scoring the root symptoms on a differential set of six pea genotypes. Eleven virulence types were characterised. The virulence type I, previously described as virulent on the whole set, was predominant and included the most aggressive isolates of all geographical origins. The other types were much less prevalent, existing as one to five isolates. Three virulence types (III, IV and V) contained no French isolates. The type III, avirulent on MN313, was composed of American isolates only, and resembled the major group recently described in the USA. A wide range of aggressiveness was found within the virulence type I, and the French isolates appeared globally more aggressive than the foreign isolates. These findings indicate that isolates from the virulence type I should be used as references in breeding programs, and that pea lines PI180693 and 552 may be the most interesting resistance sources to date, despite their only partial resistance.     

Real-time PCR identification and detection of Fuscoporia torulosa in Quercus ilex

Fuscoporia torulosa is the causal agent of white alveolar wood decay on several species including a large number of forest trees. Early detection of the fungus is essential to identify diseased trees before spread occurs to healthy plants. However, current detection methods based on isolation from infected tissues on semi-selective media are laborious, time consuming and require expertise in identifying the pathogen after isolation. In the present study, a rapid and reliable Scorpion-PCR based molecular method to identify and detect F. torulosa in planta was developed in a highly polymorphic portion of the internal transcribed spacer (ITS) regions. Specificity of primers and probe was assessed by means of both BLAST analyses and by using genomic DNA from 131 F. torulosa isolates and 43 other fungi and oomycetes from different hosts and geographic areas. In Scorpion-PCR the limit of detection was 1 pg of total DNA and a high correlation ( r ² = 0·996) was achieved between target DNA quantity and cycle threshold (Ct). Real-time PCR combined with effective procedures for DNA extraction enabled the detection of F. torulosa from naturally infected tissues of oaks with and without fruit bodies in approximately 6 h. Comparative testing showed that detection of F. torulosa in wood samples is more sensitive and reliable with real-time PCR than with conventional isolation.     

土壤中生防菌粉红粘帚霉67-1的荧光定量PCR检测方法

为了建立重要植病生防真菌粉红粘帚霉67-1菌株的荧光定量PCR检测方法,收集了目标菌株、粘帚霉属其它多个种及近缘木霉属的多个种等共18个菌株,并进行了ITS区测序。以200bp左右差异较大区段设计出探针和引物。该引物及探针能有效扩增目标菌株,而其它17株非目标菌株没有扩增,表明所设计的引物和探针具有高度特异性。以目标菌株的阳性克隆质粒作为标准物质,建立了标准曲线,相关系数为0．9989,且扩增效率较高（95．0％）。经过土壤样品试验,得出标准曲线相关系数为0．9979,表明所建立的粘帚霉67-1菌株荧光定量PCR检测方法合理有效、快速实用,适合生态学研究的要求。     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.