Development and use of a PCR assay to detect Rhizoctonia cerealis, the cause of sharp eyespot in wheat

Random amplified polymorphic DNA assays were carried out on a range of isolates of Rhizoctonia cerealis to identify markers common to all isolates. Two fragments were isolated, cloned and used to probe Southern blots of DNA from R. cerealis and isolates from a range of anastomosis groups of Rhizoctonia solani. The two fragments hybridized specifically to DNA of R. cerealis and not to DNA of any of the isolates of R. solani. Both fragments were partially sequenced and two pairs of primers were generated for use in the polymerase chain reaction (PCR). Amplification of a fragment of the anticipated size occurred following PCR of all isolates of R. cerealis and not from any of a range of fungal species associated with disease of the stem base of cereals. The primer pairs for R. cerealis were also used, along with those for Microdochium nivale and W and R-type of Pseudocercosporell herpotrichoides , to deled these pathogens in extracts from field-grown wheat plants exhibiting symptoms of sharp eyespot, eyespot, foot rot or a combination of the diseases. No relationship was found between visual disease assessment of sharp eyespot at growth stage 37 and the results of PCR However, the results of PCR and visual disease assessment at growth stage 75 were similar, indicating that visual disease assessment may not be reliable until later growth stages. This system offers the potential to detect the presence of R. cerealis in cereals and avoid problems commonly associated with conventional diagnosis of this disease and isolation of the pathogen.     

河南省首次发现梨孢镰刀菌引起的小麦赤霉病

2015年从河南省田间小麦赤霉病病穗上分离得到一种生长速度较慢的镰刀菌, 通过形态学和分子鉴定明确其分类地位, 通过田间单小花滴注法和喷雾法接种测定其致病力, 并通过高效液相色谱串联质谱分析对麦穗中的毒素种类进行测定, 明确其产毒特征?结果表明:分离得到的8个菌株均为梨孢镰刀菌, 在马铃薯葡萄糖琼脂(PDA)培养基上为白色菌落, 菌落底部产生少量红色色素, 平均生长速度为13.3 mm/d; 小型分生孢子为椭球形葡萄状, 平均大小为7.1 μm×5.8 μm, 未见大型分生孢子和厚垣孢子; 致病力弱, 且不侵染穗轴, 单小花滴注法接种条件下平均病级为0.1, 喷雾法接种条件下平均病小穗率为6.5%; 供试的8个镰刀菌菌株均不产生T-2和HT-2毒素, 均产生雪腐镰刀菌烯醇(NIV)毒素, NIV毒素含量水平为371.74~5 282.80 μg/kg, 其中3个菌株产生少量脱氧雪腐镰刀菌烯醇(DON)毒素(86.13~227.22 μg/kg)?     

Patterns of Splash Dispersed Conidia of Fusarium poae and Fusarium culmorum

Splash dispersal of Fusarium culmorum and Fusarium poae spores was studied, using inoculated straw placed on tiles as the inoculum source to infect agar strips and artificially produced leaves. In addition, patterns of spread were studied with spores from inoculated artificial leaves onto agar strips. Observed patterns of spore dispersal for each species were indistinguishable, although F. culmorum produced fewer colonies than F. poae. Furthermore, spore dispersal from inoculated straw and artificial leaves were essentially identical, with one exception; colonies arose from single conidia when spread from artificial leaves, but consisted of clumps of conidia when derived from inoculated straw. Splash dispersal patterns of both species onto the upper- and undersides of artificial leaves were different. On the upperside of the leaf, most colonies were found at the tip, while on the underside of the leaf most colonies were found at the base of the leaf. This is the first time that artificially produced leaves have been used in splash dispersal experiments.     

The development of a rapid PCR assay for detection of Fusarium moniliforme

The fungus Fusarium moniliforme infects a wide range of crops throughout the world. In maize (Zea mays L.) it causes seedling blight and root, stalk, and ear rots. A simple procedure that can be used to detect infection by F. moliliforme from infected plant tissues has been developed. A F. moniliforme genomic library was prepared and used to identify the recombinant clones containing fungal DNA sequences not hybridizing with the DNA of the host plant, maize. Based on the nucleotide sequence information obtained from the F. moniliforme pUCF2 genomic clone, specific oligonucleotides were designed and used as primers for in vitro DNA amplification by the polymerase chain reaction. An amplification product was obtained with F. moniliforme DNA preparations whereas no amplified DNA was detected with DNAs from other fungal pathogens, including various Fusarium species, or from the host plant. This PCR analysis was successfully employed to identify F. moniliforme directly from the mycelia that develop from naturally infected maize seeds, with no need to obtain pure fungal cultures for reliable diagnosis. The protocol can be used for the diagnosis of infected plants and soils in epidemiological studies of Fusarium diseases, for seed health testing, and for evaluation of susceptibility to colonization in commercial maize hybrids.     

Characterization of a Fusarium poae world-wide collection by using molecular markers

Fusarium poae has been considered as a minor species among those that cause Fusarium Head Blight (FHB) disease but in recent years several researchers have documented a high frequency of occurrence of this species. In this study, a total of 173 F. poae isolates from Argentina, Belgium, Canada, England, Finland, France, Germany, Hungary, Italy, Luxembourg, Poland, Switzerland and Uruguay were evaluated by using inter simple sequence repeats (ISSR) and amplified fragment length polymorphism (AFLP) to evaluate genetic variability within F. poae and to amplify MAT idiomorphs as a possible mechanism that could explain part of the variability found in this species. The molecular analysis obtained from both molecular markers showed a high intraspecific variability. However, a partial clustering between F. poae isolates and their geographic origin was obtained by ISSR markers while AFLP showed isolates from different geographic locations distributed throughout the dendrogram. Moreover, ISSR grouped all the F. poae isolates into a different cluster from the F. langsethiae and F. sporotrichioides isolates used as outgroups compared with the dendrogram obtained using AFLP markers. Analysis of molecular variance (AMOVA) indicated a high genetic variability in the F. poae collection, with most of the genetic variability resulting from differences within, rather than between, American and European populations by using both molecular markers. Regarding MAT idiomorphs, for most F. poae isolates both MAT-1 and MAT-2 were present from each isolate.     

禾谷镰孢菌产玉米赤霉烯酮毒素zearalenone的PCR检测

玉米赤霉烯酮（zearalenone,ZEN）是由镰孢菌产生的真菌毒素,该毒素具有雌性激素生物活性。PKS4基因是禾谷镰孢菌中玉米赤霉烯酮生物合成途径中的必需基因。根据PKS4基因序列,设计一对PKS4基因特异性引物,通过检测PKS4基因的存在间接检测ZEN毒素的产生。特异引物在禾谷镰孢菌菌株以及被禾谷镰孢菌侵染的小麦籽粒中都能稳定地扩增出大小为1 076 bp的特异片段,扩增片段与PKS4基因（DQ019316）相同区段序列相似性达99.63%。同时,酶联免疫吸附法（ELISA）验证了PCR的检测结果,证明了该技术的可靠性。     

Development of a PCR test to detect the downy mildew causal agent Plasmopara halstedii in sunflower seeds

Plasmopara halstedii , the causal agent of downy mildew of sunflower, is an obligate parasite but viable sporangia and oospores of the pathogen may be found in a quiescent state in seeds of sunflower and therefore may be transported with sunflower seeds in international commercial exchanges. In order to prevent the spread of this pathogen, especially the introduction of potentially new races, an efficient method to analyse sunflower seed samples is required. In this study, a P. halstedii -specific polymerase chain reaction (PCR) test was developed based on the ribosomal large sub unit (LSU) DNA. The forward (PHAL-F) and reverse (PHAL-R) PCR primers were designed from two polymorphic regions of LSU. After screening 22 isolates of P. halstedii corresponding to different races and countries and 32 other oomycete, deuteromycete and ascomycete isolates, the PHAL-F/R primers amplified only a single PCR band of c. 310 bp from P. halstedii . The PHAL-F/R PCR test could detect as little as 3 pg of P. halstedii genomic DNA per 20  µ L reaction volume and enabled the direct detection of P. halstedii in 35 g sunflower seed samples without the need for any prior biological baiting step. An internal amplification control (IAC) was developed to help discriminate against false negative samples due to the potential presence of inhibitory compounds in DNA extracts. The test was successfully used on samples of naturally contaminated seeds. These new molecular tools should be of great interest for quarantine seed testing purposes.     

Variability amongst strains of Fusarium poae assessed by vegetative compatibility and RAPD polymorphism

Vegetative compatibility tests and random amplification of polymorphic DNA (RAPD) were used to assess genetic relationships amongst 54 strains of Fusarium poae obtained from various geographical regions. Twenty-seven strains were assigned to eight multiple member vegetative compatibility groups (VCGs), while the other 27 isolates were found to form single-member VCGs. There was a partial correlation between VCG and geographical origin, but the relationship was not always clear. However, no correlation was observed between the VCG and the host plant of origin. RAPD patterns were closely associated with VCGs in all cases. Members of VCGs that were interconnected by bridging isolates formed common branches in the phenogram constructed on the basis of the RAPD patterns, while strains that belonged to single-member VCGs were scattered throughout the phenogram. These data demonstrate that the combination of traditional and molecular methodologies allows reliable intraspecific subdivisions in an asexual fungus, which is a secondary invader of a wide range of host plants, and so has never been subject to the intense selection pressure of a single host species and lacks pathogenic subgroups.     

Components of partial disease resistance detected using a detached leaf assay in CIMMYT Fusarium head blight resistant wheat germplasm

One hundred and nineteen entries from the CIMMYT International Wheat and Maize Improvement Centre 2004/05 Fusarium head blight (FHB) resistance screening nursery were evaluated as possible sources of novel components of partial disease resistance (PDR), against FHB and Microdochium nivale snow mould, detected using a detached leaf assay. In addition the FHB resistant cvs Arina, Alsen and Frontana and 21 European wheat genotypes were included for comparison. There was wide variation among CIMMYT entries for the PDR components incubation period, latent period and lesion length (P < 0.001) and European lines for incubation and latent periods (P < 0.001). The CIMMYT entries with the longest latent periods were not superior to cv. Arina, the best European source of this PDR component identified to date. Notably the CIMMYT lines exhibiting the longest latent periods had Aegilops squarrosa (878) in their pedigree, indicating that Ae. squarrosa (878) may be a source of enhanced resistance detected by latent period. Macroscopic observation suggested that the underlying mechanisms contributing to latent period may differ among the CIMMYT germplasm and European sources of long latent period such as cv. Arina. Among the CIMMYT germplasm, incubation period was only weakly correlated with latent period (r = 0.25; P < 0.01); this also was the case among European genotypes (r = 0.36; P < 0.05) supporting previous findings that these PDR components are largely under separate genetic control. However, the correlation was higher on a subset of the most resistant and susceptible lines for latent period (r = 0.73 and r = 0.44; incubated at 10 °C and 15 °C, respectively). While a number of the European lines had latent periods that were comparable to cv. Arina many were significantly shorter indicating potential for improvement in this PDR component. Adaptations to the experimental design utilized in the present experiments for the efficient evaluation of large numbers of genotypes utilizing the detached leaf assay are discussed.     

Real-time PCR assay for quantification of Tilletia caries contamination of UK wheat seed

A quantitative real-time PCR assay using TaqMan chemistry has been developed to quantify the level of Tilletia spp. contamination in wheat-seed lots. In the UK wheat seed is predominantly contaminated with Tilletia caries (syn. Tilletia tritici ), and the probability of detecting other Tilletia spp. is negligible. DNA standards, prepared from T. caries spores, were calibrated using a set of 26 seed samples, with T. caries contamination levels ranging from 0 to 1000 spores per seed. The linear calibration model obtained by the regression of log₁₀ (number of spores per seed + 1) on mean log₁₀ DNA ( µ g) produced a coefficient of determination ( R ²) of 0·904. The calibration model was tested using 226 seed samples; of these, 91% fell within the 95% confidence intervals. Of the 21 samples that were outside the limits, 16 were overpredictions and five underpredictions. The five underpredictions were all from seed samples where contamination was less than one spore per seed. The model predicts that samples with 44 pg of DNA will be below one spore per seed with 95% probability. Of the 226 test samples compared with this threshold, 99 contained less than 44 pg DNA, and these were found to have less than one spore per seed by microscopic assay. This real-time assay allows an increase in test throughput and provides the sensitivity required for an advisory threshold of one spore per seed.     

Detection and quantification of Fusarium oxysporum f. sp. cucumerinum in environmental samples using a specific quantitative PCR assay

The rapid and reliable identification and quantification of pathogens is essential for the management of economically important plant diseases. Fusarium oxysporum f. sp. cucumerinum is the soil borne fungus responsible for Fusarium vascular wilt of cucumber. In this study, we report the development of a specific and reliable real-time quantitative PCR assay and the development of an ultra-sensitive diagnostic pseudo-nested PCR assay. The capacity of the PCR assays to accurately identify and quantify Fusarium oxysporum f. sp. cucumerinum was experimentally tested by the development of standard curves from serial dilutions of copy numbers in a range of complex environmental DNA samples. The amplification efficiency, sensitivity and reproducibility of the qPCR assays were not significantly affected by the presence of any of the non-target background DNA tested. In quantitative real-time PCR, as few as 100 copies could be reliably quantified, and in simple and pseudo-nested PCR as little as 10 pg and 10 fg, respectively, could be detected. This rapid and sensitive qPCR method can be used to facilitate investigations into plant–pathogen interactions, epidemiology, and disease management practices.     

基于多重PCR技术的西瓜噬酸菌分组检测方法及其应用

瓜类细菌性果斑病是瓜类作物上重要的种传细菌性病害,其病原菌西瓜噬酸菌Acidovorax citrulli为我国进境植物检疫性有害生物,种子种苗带菌是病害长距离传播的重要来源,种子种苗的快速检测对病害综合防控具有重要的意义。根据寄主的差异西瓜噬酸菌分为两个组,Ⅱ组菌株比Ⅰ组菌株具有更高的铜制剂敏感性,因此,西瓜噬酸菌的分组检测可为病害田间防治中铜制剂的精准使用提供科学依据,避免化学农药的过量施用。本研究筛选了现有的西瓜噬酸菌种间特异性引物和种内分组特异引物,建立并优化了一个多重PCR体系,实现了通过一步试验,就能够准确将西瓜噬酸菌与近缘种和其他植物病原细菌区分开来,并直接鉴定到西瓜噬酸菌不同组。该多重体系可以从带菌种子浸泡液和感病植物组织研磨液中直接检测到西瓜噬酸菌的不同组,且稳定性好,具有应用于生产实践的潜力。     

Development of a multiplex PCR for the identification of Fusarium solani and F. oxysporum in a single step

Journal of Plant Diseases and Protection - Fusarium solani and F. oxysporum are plant pathogenic fungi that cause root rot and wilt, respectively, in many economically important crops....     

水稻干尖线虫快速分子检测技术研究

根据水稻干尖线虫的rDNA-ITS序列,设计出水稻干尖线虫的特异性引物,利用PCR技术对水稻干尖线虫(Aphelenchoides besseyi)部分rDNA-ITS1和部分5.8S基因核苷酸序列进行特异性扩增。实现了单条活的或4%的甲醛(FG)固定的水稻干尖线虫的快速检测。     

Early PCR-based detection of Fusarium culmorum,F. graminearum,F. sporotrichioides and F. poae on stem bases of winter wheat throughout Poland

Foot rot and crown rot are fungal diseases of wheat caused by a complex of Fusarium species. They have a huge economic impact mainly due to yield reduction. A survey was conducted to identify four Fusarium species, occurring on wheat stem bases, using species-specific PCR assays in samples collected during spring of 2012. The dominant species was F. graminearum, which was identified in above 64 % of samples. F. culmorum was detected in 15.71 %, F. poae in 15.71 % and F. sporotrichioides in 5.71 % wheat fields. Most of the wheat fields in the eastern Poland were infected with at least one or two of Fusarium species, while in central Poland no Fusarium species were identified in most of the fields. The presence of F. graminearum tends to favor the presence of F. culmorum and this effect was visible also for F. poae and F. sporotrichioides. The frequency of F. graminearum and F. culmorum detections were highest where wheat crops were preceded by maize and in the samples from late sown fields. The opposite observation was made for F. poae and F. sporotrichioides, where the number of detections of these species was higher in samples from early sown fields. The number of detected Fusarium species was significantly lower in samples collected from fields protected with autumn herbicide in comparison to unprotected fields. The rate of autumn N fertilization did not affect the number of Fusarium detections.     

Influence of temperature,humidity duration and growth stage on the infection and mycotoxin production by Fusarium langsethiae and Fusarium poae in oats

High occurrence of Fusarium poae (FP) and Fusarium langsethiae (FL) and their mycotoxins nivalenol (NIV) and T-2/HT-2 have been observed in Swiss oats. Early prediction of mycotoxin levels is important for farmers and the cereal industry to minimize the risk of contaminated food and feed. Therefore, climate chamber experiments were conducted to investigate the influence of different temperatures (10, 15, 20 °C) and durations (4, 8, 12 h) at 99% relative humidity (RH) on the infection of oats with FP and FL. In addition, to discover the most susceptible period of oats, artificial FL inoculations were conducted at different growth stages. Field experiments were performed to observe the dispersal of these fungal species within the field and to investigate the weather conditions that influence the dispersal. The climate chamber experiments revealed higher contamination with NIV and T-2/HT-2 in the 10 °C treatments and with a prolonged humidity duration of 12 h 99% RH. Inoculations of oat plants at early (DC 61) and mid (DC 65) anthesis, led to higher FL infection and T-2/HT-2 accumulation in the grains compared with treatments at earlier growth stages, which might be due to an increased susceptibility during anthesis. No indication for spore dispersal was observed in the field experiments. The results obtained, together with the cropping factors that influence infection and mycotoxin production, could be used as a first step in developing forecasting models to predict the contamination of oats with the mycotoxins NIV and T-2/HT-2.