PCR-based Assays to Assess Wheat Varietal Resistance to Blotch (Septoria Tritici and Stagonospora Nodorum) and Rust (Puccinia Striiformis and Puccinia Recondita) Diseases

A multiplex Polymerase Chain Reaction (PCR) assay was developed to detect and quantify four fungal foliar pathogens in wheat. For Septoria tritici (leaf blotch) and Stagonospora nodorum (leaf and glume blotch), the -tubulin gene was used as the target region. Diagnostic targets for Puccinia striiformis (stripe or yellow rust) and P. recondita (brown rust) were obtained from PCR products amplified with -tubulin primer sequences. Final primer sets were designed and selected after being tested against several fungi, and against DNA of infected and healthy wheat leaves. For detection of the four pathogens, PCR products of different sizes were amplified simultaneously, whereas no products were generated from wheat DNA or other non-target fungi tested. The presence of each of the diseases was wheat tissue- and cultivar specific. Using real-time PCR measurements with the fluorescent dye SYBR Green I, PCR-amplified products could be quantified individually, by reference to a standard curve generated by adding known amounts of target DNA. Infection levels for each of the diseases were measured in the flag leaf of 19 cultivars at Growth Stage (GS) 60–64 in both 1998 and 1999. The infection levels for the cultivars were ranked, and showed, with a few exceptions, a good correlation with the NIAB Recommended List for winter wheat, which is based on visual assessment of symptoms. With PCR, the presence of the different pathogens was accurately diagnosed and quantification of pre-symptomatic infection levels was possible. Although sampling and DNA detection methods need further optimisation, the results show that multiplex PCR and quantitative real-time PCR assays can be used in resistance screening to measure the interaction between different pathogens and their hosts at different growth stages, and in specific tissues. This should enable an earlier identification of specific resistance mechanisms in both early-stage breeding material and field trials.     

Conventional PCR and real time quantitative PCR detection of <Emphasis Type="Italic">Phytophthora cryptogea</Emphasis> on <Emphasis Type="Italic">Gerbera jamesonii</Emphasis>

A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 10³ P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.     

PCR assays for the sugarcane rust pathogens Puccinia kuehnii and P. melanocephala and detection of a SNP associated with geographical distribution in P. kuehnii

Puccinia kuehnii and P. melanocephala cause orange and brown rust of sugarcane, respectively. Puccinia kuehnii has been confirmed in Asia, Australia and recently, the Caribbean basin, whereas P. melanocephala is distributed among the majority of sugarcane growing regions. Differentiating these two economically significant pathogens visually is problematic and limited to material exhibiting mature disease symptoms or spores. Partial ITS1, ITS2 and complete 5·8S sequences were generated from P. kuehnii and P. melanocephala isolates from around the world. PCR primers and dual labelled hydrolysis probes were designed for each pathogen for use in real‐time PCR and optimized using locked nucleic acids (LNA). The primers amplified DNA from their target pathogens and not from other species of Puccinia or fungal species isolated from sugarcane leaves. Optimized real‐time PCR conditions allowed the detection of 0·19 pg of P. kuehnii or P. melanocephala genomic DNA and differentiated the pathogens on sugarcane leaves prior to observing typical symptoms in the field. Primer‐introduced restriction analysis‐PCR (PIRA‐PCR) was used to detect a single nucleotide polymorphism (Pk ITS1 183A>G) in ITS1 of P. kuehnii. Allele 183A was observed in all samples, whereas 183G was detected in 52% of samples from Asia and Australia yet absent from all Caribbean basin samples. Long distance spore dispersal, dispersal through an intermediate location or improper movement of contaminated material could explain the introduction of P. kuehnii to the Western hemisphere. However, the current proliferation of the pathogen in the Americas is limited to isolates which contain only the 183A allele.     

西藏小麦条锈菌群体结构与遗传多样性

为查明西藏小麦条锈菌Puccinia striiformis f. sp. tritici群体结构和遗传多样性,采用中国鉴别寄主和近等基因系鉴别寄主,以及竞争性等位基因特异性PCR-单核苷酸多态性（kompetitive al-lele specific PCR-single nucleotide polymorphism,KASP-SNP）分子标记对2017年采自西藏的150个小麦条锈菌菌系分别进行表型分析和基因型分析。表型分析结果显示,中国鉴别寄主将150个菌系区分为 12 个已知小种、6 个已知致病类型和 13 个未知致病类型,所有菌系均不能侵染中四和Triticum spelta album鉴别寄主。近等基因系鉴别寄主将150个菌系区分为88个毒性类型,这些毒性类型均不侵染携带抗性基因Yr5、Yr10或Yr15的品种。基因型分析结果显示,26对引物将150个菌系划分为73个基因型,表明西藏小麦条锈菌群体基因型丰富。基因流分析结果表明,波密县与洛扎县小麦条锈菌亚群体之间的基因流N_m最高,达5.86,米林县西部与波密县、洛扎县、巴宜县、米林县东部条锈菌亚群体之间的N_m较低,分别为0.25、0.34、0.42和0.67,表明西藏不同地区条锈菌群体之间基因交流强度差异较大。说明西藏作为我国小麦条锈病的独立流行区,条锈菌群体毒性结构复杂,遗传多样性高。     

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.     

河南省小麦全蚀病菌的鉴定、进化分析及标志基因筛选

为了解河南省郑州市中牟市小麦全蚀病菌的变种类型及进化情况,筛选全蚀病菌侵染小麦的分子标记,采用形态学观察、科赫氏法则验证、ITS序列分析和系统进化树构建等方法对分离的小麦全蚀病菌进行鉴定,并对其侵染后小麦病原相关蛋白(pathogen-related protein,PR)基因的表达进行实时定量PCR分析。结果表明,显微形态观察初步确定分离的小麦全蚀病菌为禾顶囊壳Gaeumannomyces graminis(Sacc.)v. Arx.Olivier,经科赫氏法则验证、ITS序列比对及系统进化树分析进一步证明该病菌均为禾顶囊壳。本研究分离的小麦全蚀病菌与地域相距较远的英国、美国的小麦全蚀病菌间的同源性比与来自中国陕西省的小麦全蚀病菌的同源性更高。采用禾顶囊壳4个变种的特异性引物进行PCR扩增,所有分离病菌均扩增出了小麦变种的特异性条带,证实分离菌株为禾顶囊壳小麦变种G. graminis var. tritici。实时定量PCR分析发现,病原相关蛋白基因PR4a、PR4b、PR2、PR10受到小麦全蚀病菌侵染后表达量均显著上调,在侵染后第5～6天达到最高峰,表明这些基因可作为小麦全蚀病菌侵染小麦的标志性基因。     

Analysis of population structure of <Emphasis Type="Italic">Puccinia striiformis</Emphasis> in Yunnan Province of China by using AFLP

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most destructive wheat diseases in China. Yunnan Province, located in south-western China, possesses unique features of geography, climate, wheat growth and stripe rust epidemics, different from main epidemic regions in China. The isolates of this pathogen were collected from nine counties in Yunnan Province during February to May of 2008. Used as a comparison, isolates were also collected from five counties of Gansu Province, the province important in inter-regional stripe rust epidemics in China. Amplified fragment length polymorphism (AFLP) method was applied to study the population genetics of the pathogen among different populations in these two provinces. Forty one AFLP genotypes were obtained from 150 isolates and the genotype qj3 showed the highest frequency in Yunnan Province. While 22 genotypes were detected from 40 isolates, no genotype showing as predominant was identified in Gansu Province. Genotypic diversity in Gansu Province was higher than that in Yunnan Province. A free recombination signature was detected in Gansu Province but not in Yunnan Province. We concluded that the population of P. striiformis in Yunnan Province can be considered as a clonal population.     

Temperature adaptation in Australasian populations of Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the major fungal pathogens of wheat. A new pathotype was introduced to Australia in 2002 and several derivative pathotypes were detected in subsequent seasons. It has been suggested that the severity of stripe rust outbreaks in Australia since 2002 could be as a result of traits other than virulence in the pathogen population. This study was conducted to investigate the hypothesis that the stripe rust pathogen population dominant in Australia since 2002 was better adapted to warm temperature conditions compared to previous pathogen populations. Sixteen pathotypes were selected to examine the influence of two contrasting temperature regimes during the 24 h incubation (10°C and 15°C) and the subsequent post‐inoculation (17°C and 23°C) periods on latent period and infection efficiency on four susceptible wheat cultivars. In addition, the effect of two contrasting incubation temperatures on urediniospore germination was examined. The results indicated that pathotypes of P. striiformis f. sp. tritici detected after 2002 did not show evidence of adaptation to high temperatures, which suggests that other factors contributed to the observed increased aggressiveness.     

基于Logistic、IBk以及Randomcommittee方法的条锈病潜育期小麦冠层光谱的定性识别

为寻求在小麦条锈病潜育期能探知和监测病害的简单便捷方法,通过人工接种不同品种小麦诱发条锈病,在小麦条锈病菌尚处于潜育期时,采集小麦冠层光谱数据,并利用双重Real-time PCR分子生物学技术检测条锈病菌潜育菌量,基于Logistic、IBK以及Randomcommittee三种方法,在不同建模比、不同参数变换下建立可识别潜育期小麦条锈病的数学模型。结果表明,在全波段范围内(325~1 075 nm),3种方法所建模型模拟识别潜育期小麦条锈病是可行的,但识别效果有一定差异,基于Logistic、IBK以及Randomcommittee方法所建模型的平均准确率分别为83.95%~84.51%、87.72%~88.98%、93.19%~93.46%。因此,基于Randomcommittee方法所建模型的识别准确率最高,效果最好,更适合小麦条锈病潜育期的定性识别。     

A Phytophthora conserved transposon‐like DNA element as a potential target for soyabean root rot disease diagnosis

A transposon‐like element, A3aPro, with multiple copies in the Phytophthora sojae genome, was identified as a suitable detection target for this devastating soyabean root rot pathogen. The PCR primers TrapF1/TrapR1 were designed based on unique sequences derived from the transposon‐like sequence. A 267‐bp DNA fragment was amplified using this primer pair, the specificity of which was evaluated against 118 isolates of P. sojae, 72 isolates of 25 other Phytophthora spp., isolates of Pythium spp. and isolates of true fungi. In tests with P. sojae genomic DNA, detection sensitivities of 10 pg and 10 fg DNA were achieved in standard PCR (TrapF1/TrapR1) and nested PCR (TrapF1/TrapR1 and TrapF2/TrapR2), respectively. Meanwhile, PCR with TrapF1/TrapR1 primers detected the pathogen at the level of a single oospore, and even one zoospore. These primers also proved to be efficient in detecting pathogens from diseased soyabean tissues, residues and soils. In addition, real‐time quantitative PCR (qPCR) assays coupled with the TrapF1/TrapR1 primers were developed to detect and quantify the pathogen. The results demonstrated that the TrapF1/TrapR1 and TrapF2/TrapR2 primer‐based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in plants and in production fields.     

模拟酸雨对小麦条锈病流行学组分的影响

为明确酸雨对小麦条锈病菌孢子萌发及病害流行学组分的影响,采用不同酸度的模拟酸雨(p H值分别为2.5、3.5、4.5和5.6)处理小麦条锈病菌Puccinia striiformis f.sp.tritici生理小种CYR32和CYR33的夏孢子,并将不同酸度模拟酸雨处理后的夏孢子接种小麦感病品种铭贤169,采用组分分析法研究酸雨对病害发展过程的影响。结果表明,随着模拟酸雨酸度的增强,生理小种CYR32和CYR33的夏孢子萌发率均显著降低,萌发24 h后对照组生理小种CYR32和CYR33的夏孢子萌发率分别为93.7%和79.8%,在p H 3.5模拟酸雨处理后萌发率仅为13.1%和8.6%。温室内接种试验结果显示,不同酸度模拟酸雨对生理小种CYR32和CYR33的侵染概率、潜育期和病情指数均有显著影响,对生理小种CYR32和CYR33病斑扩展率影响不显著;与对照相比,p H 3.5模拟酸雨处理后生理小种CYR32和CYR33的侵染概率分别下降79.9%和79.8%。结果表明强酸雨(p H 3.5)显著延长小麦条锈菌潜育期,减少病斑产孢量,降低病情指数和病害进展曲线下面积。     

Gene-based analysis of <Emphasis Type="Italic">Puccinia</Emphasis> species and development of PCR-based marker to detect <Emphasis Type="Italic">Puccinia striiformis</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis> causing yellow rust of wheat

Stripe rust is considered as the current major rust disease affecting winter cereal production across the world. A quick, reliable PCR-based marker was developed here to detect, identify and rapidly monitor Puccinia striiformis f. sp. tritici (Pst) in wheat-growing areas. Three respective sets of primers, designed from β-tubulin, squalene monooxygenase and ketopantoate reductase genes selected from the full genome of Puccinia striiformis f. sp. tritici, amplified sequences of 239, 358 and 1518 bp, respectively, in Pst pathotypes. A fragment of 1518 bp unique to Pst pathotypes was amplified using primer set PstKeto F1_30/Pst KetoR1_1547 and distinguished the pathogen clearly from different Puccinia spp. and other fungal pathogens. The detection limit of the marker (KetoPstRA₁₅₀₀, accession no. KU240073) by conventional PCR assay was 10 pg. This marker could detect the pathogen in the host before symptom expression. The sensitivity and utility of the marker were further enhanced in a qPCR-based assay that was developed with a newly designed primer set PstKeto F1_1246/Pst KetoR1_1547, which amplified a product of 302 bp and detected as little as 10 fg of DNA. This PCR/qPCR based marker is suitable for studying cultivar resistance, which requires accurate quantification of the pathogen in diseased host tissue.     

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.     

Hyperspectral signal decomposition and symptom detection of wheat rust disease at the leaf scale using pure fungal spore spectra as reference

This study establishes a method to detect and distinguish between brown rust and yellow rust on wheat leaves based on hyperspectral imaging at the leaf scale under controlled laboratory conditions. A major problem at this scale is the generation of representative and correctly labelled training data, as only mixed spectra comprising plant and fungal material are observed. For this purpose, the pure spectra of rust spores of Puccinia triticina and P. striiformis, causal agents of brown and yellow rust, respectively, were used to serve as a spectral fingerprint for the detection of a specific leaf rust disease. A least-squares factorization was used on hyperspectral images to unveil the presence of the spectral signal of rust spores in mixed spectra on wheat leaves. A quantification of yellow and brown rust, chlorosis and healthy tissue was verified in time series experiments on inoculated plants. The detection of fungal crop diseases by hyperspectral imaging was enabled without pixel-wise labelling at the leaf scale by using reference spectra from spore-scale observations. For the first time, this study shows an interpretable decomposition of the spectral reflectance mixture during pathogenesis. This novel approach will support a more sophisticated and precise detection of foliar diseases of wheat by hyperspectral imaging.     

Genetic diversity of Puccinia striiformis from cereals in Alberta,Canada

Stripe rust of wheat caused by Puccinia striiformis f. sp. tritici has recently become a production problem on wheat in Alberta, Canada, and stripe rust of barley caused by P. striiformis f. sp. hordei occurs regularly. A total of 261 isolates of P. striiformis were collected from wheat, barley, Hordeum jubatum and triticale plants in Alberta, Canada from 2007 to 2012, and compared to isolates from other provinces and the USA. The genetic diversity of the pathogen was assessed using 11 simple sequence repeat (SSR) markers and by examining a length polymorphism in the ribosomal DNA (rDNA) intergenic spacer 1 (IGS1) region. A total of 28 SSR genotypes were detected within Alberta. The 13 genotypes common on wheat (P. striiformis f. sp. tritici) were distinct from the 15 genotypes common on barley (P. striiformis f. sp. hordei). Four SSR genotypes, two within each forma specialis, represented 85% of the isolates recovered. Genotypic diversity was low, population genetic analysis indicated a clonal structure, and the genotypes were widely dispersed. In both formae speciales, the dominant genotype varied between years. The second most common P. striiformis f. sp. hordei genotype was found to be more closely related to older P. striiformis f. sp. tritici genotypes from the USA than to other P. striiformis f. sp. hordei genotypes.     

PCR-based detection of sunflower white blister rust (<Emphasis Type="Italic">Pustula helianthicola</Emphasis> C. Rost &; Thines) in soil samples and asymptomatic host tissue

Sequencing of partial cox2 (part of the mitochondrial cytochrome-c-oxydase (COX) gene) was performed with samples from the oomycete genus Pustula, the white blister rusts of Asteraceae and related families. Sequence comparison uncovered several single nucleotide polymorphisms (SNPs) between P. spinulosa and host specific strains of Pustula isolated from Senecio vulgaris, Tragopogon pratensis and cultivated sunflower, Helianthus annuus. Based on these differences, specific primers were designed for PCR-based detection of white blister rust strains pathogenic to sunflower. The specificity of the primers was confirmed by cross testing with DNA from various oomycetes occurring in the same locality. The limit of detection for DNA of P. helianthicola was 10 pg. This allowed detection with DNA from single sporangia and single oospores. The PCR-based experiments allowed detection of the presence of sunflower white blister rust in soil samples from fields on which infected plants had been cultivated several months before. Moreover, the molecular tools were successfully applied to trace the pathogen in asymptomatic tissue of infected plants, demonstrating the systemic nature of Pustula on sunflower.     

Description and molecular phylogeny of <Emphasis Type="Italic">Ditylenchus gilanicus</Emphasis> n. sp. (Nematoda: Anguinidae) from northern forests of Iran

Commercial areas containing Eucalyptus plantations have expanded in recent years due to increased demands for pulp, paper and bioenergy. One of the threats that can reduce Eucalyptus production is the eucalyptus rust disease caused by Austropuccinia psidii, a biotrophic fungus that affects a broad range of Myrtaceae. An accurate diagnosis tool for the early detection of rust disease could be useful in breeding programs for selection of resistant plants against rust, in phytosanitary purposes or in rust epidemics studies. The aim of the present work was to develop a SYBR Green-based quantitative real-time PCR (qPCR) assay for the early detection and quantification of A. psidii in Eucalyptus grandis leaves. Three sets of primers based on the A. psidii ribosomal DNA intergenic space region (IGS), beta-tubulin and elongation factor genes were designed and evaluated. The assays using the IGS primer set resulted in the highest detection efficiency, detecting a lower limit of 0.5 pg of A. psidii DNA. Under artificial inoculation in plants, A. psidii was detected immediately after pathogen inoculation until 240 h post-inoculation using qPCR. In field validation of the method, A. psidii was detected using qPCR in naturally infected leaves with or without rust symptoms. This easy and fast method can be used for an efficient detection of A. psidii in E. grandis leaves. The implications of this tool for rust studies are discussed below.     

Direct detection of <Emphasis Type="Italic">Taphrina deformans</Emphasis> on peach trees using molecular methods

The ascomycetous fungus Taphrina deformans is the agent of peach leaf curl, a worldwide disease of peach potentially devastating to both crop yields and tree longevity. Conspicuous leaf curl symptoms result from the invasion of host tissue by the strictly parasitic mycelial phase of the T. deformans dimorphic life-cycle. Successful isolation of the fungus in pure culture is cumbersome and limited to late spring/early summer (time of ascospore discharge from infected leaves) and only rarely has the asymptomatic yeast phase been isolated from buds. Molecular methods, namely those based on the hybridisation of nucleic acids, are advantageous for diagnostic purposes since they do not require isolation of the fungus on culture media. Direct amplification using the polymerase chain reaction (PCR) and fluorescent in situ hybridisation (FISH) were tested for diagnosis of peach leaf curl disease in order to provide a fast and reliable method for disease risk assessment. Specific primers and probes were designed based on available ribosomal DNA sequence data. Positive and specific diagnoses of peach leaf curl were achieved with primer TDITS1, using PCR-detection, and probe TDE634, using FISH, both on infected leaves and in washings of asymptomatic peach buds.     

PCR‐based identification of Pythium spp. causing cavity spot in carrots and sensitive detection in soil samples

On the basis of ITS sequences PCR primers were designed for the identification of the five Pythium species found to be most important for the development of carrot cavity spot in Norway: P. intermedium, P. sulcatum, P. sylvaticum, P. violae and P. ‘vipa’. The P. ‘vipa’ isolates had a unique ITS sequence, differed morphologically from all other Pythium isolates, and thus probably represent a new species. The PCR primers were species‐specific with no cross‐reaction to other Pythium species or to fungal isolates from carrot tested. The detection limits varied for the different primer pairs. The two most sensitive assays allowed detection of as little as 5 fg DNA. All five Pythium species could be detected in lesions from diseased carrots. Weak positive signals were obtained from some carrot samples without symptoms. PCR assays allowed detection of pathogens in soil. In samples of soil known to produce cavity spots on cropped carrots, strong signals were obtained. In several soil samples more than one of the five Pythium species could be detected. The utilization of this diagnostic PCR assay in analysis of field soil and carrot tissue might in the future be exploited to reduce the incidence of this serious carrot disease.     

小麦条锈病生防菌株的筛选、鉴定及其防治效果

为获得对小麦条锈病具有生防潜力的菌株,通过土壤稀释法和皿内拮抗法分离筛选对小麦条锈菌Puccinia striiformis f.sp.tritici（Pst）有良好拮抗作用的菌株,基于形态学观察、生理生化特性和16S rDNA系统发育分析对其进行分类鉴定,测定其抑菌活性、抑菌谱及生防特性,并通过盆栽试验进一步验证其生防潜力。结果显示,筛选出1株菌株XH可以显著抑制Pst夏孢子萌发,结合形态学和分子生物学特征将其鉴定为直丝紫链霉菌Streptomyces rectiviolaceus;菌株XH发酵滤液对Pst夏孢子的萌发抑制率为94.36%,致死率达91.53%,且该菌株对测试的7种常见植物病原菌均有抑制作用,可以分泌淀粉酶、蛋白酶、纤维素酶和嗜铁素,具有固氮作用;盆栽试验结果显示,菌株XH的菌体悬浮液和发酵滤液可显著降低小麦叶片的Pst侵染量,对小麦条锈病的防治效果分别为54.26%和67.22%。表明菌株XH作为小麦条锈病生防菌株具有较好的开发应用潜力。