Relationship between brown foot rot and DNA of Microdochium nivale, determined by quantitative PCR, in stem bases of winter wheat

Relationships between the incidence and severity of brown foot rot and of pathogenic fungi, determined by diagnostic and quantitative PCR, were investigated during the growth of nine winter wheat crops in three cropping seasons. Microdochium nivale vars nivale and majus were the only brown foot rot pathogens present in significant amounts. Relationships between disease symptoms and amounts of pathogen DNA were often weak in early spring (when shoot-base symptoms are usually most difficult to ascribe to particular pathogens by visual examination) because of indistinct symptoms and small amounts of pathogen. Relationships were strongest during stem elongation. The amount of M. nivale in the tissues tended to decline in the summer as the plants matured, apparently disappearing partially from necrotic lesions to which it contributed, resulting in a weakened relationship between symptoms and pathogen DNA. Regression analyses of brown foot rot on amounts of M. nivale DNA for different wheat cultivars generally produced lines with similar slopes but were often most significant for the cultivar with most eyespot resistance (i.e. with least confounding eyespot) or most apparently genuine brown foot rot. DNA of Fusarium spp. was rarely present in amounts sufficient to quantify.     

Development of Stem-base Pathogens on Different Cultivars of Winter Wheat Determined by Quantitative PCR

The progress of development of stem-base pathogens in crops of second winter wheat was plotted in nine experiments in three years. The amount of each pathogen present was determined by quantitative PCR. Where Tapesia yallundae was present in quantifiable amounts, it usually developed earlier than the other eyespot pathogen, T. acuformis. Both species were usually present in greater amounts on cultivars which are more susceptible to eyespot. The sharp eyespot pathogen, Rhizoctonia cerealis, developed more erratically than either of the Tapesia spp. and there were no consistent effects on different cultivars. Fusarium spp., the cause of brown foot rot, were rarely present in quantifiable amounts, but Microdochium nivale was usually present as one or both of the varieties nivale and majus. Late-season (after anthesis) decreases in M. nivale suggest that any brown foot rot symptoms attributable to this fungus would have fully developed earlier. Cultivar differences in amounts of M. nivale were most clear in stems during internode extension and when relatively large amounts of DNA were present. Such differences approximately reflected eyespot susceptibility, cv. Soissons containing most and cv. Lynx containing least DNA. The results emphasise the difficulty in relating diagnoses, by quantitative PCR or other means, at early growth stages when decisions to apply fungicides against stem-base disease are made, to later disease severity.     

Visual disease and PCR assessment of stem base diseases in winter wheat

Stem base disease (eyespot, sharp eyespot and brown foot rot) was assessed visually and by the polymerase chain reaction (PCR) technique on single plants sampled at four-week intervals in two crops of winter wheat grown in the UK in 1992–3. PCR assays were conducted for Fusarium avenaceum , F. culmorum , both varieties of Microdochium nivale , both eyespot-causing species of Tapesia and Rhizoctonia cerealis . PCR diagnoses were compared with visual diagnoses at each time point. Eyespot was caused principally by T. acuformis (R-type) and developed rapidly late in the season. Visual diagnoses of eyespot were largely confirmed by PCR but T. acuformis was detected in many plants lacking eyespot symptoms. R. cerealis was detected at relatively low incidences on both sites, and sharp eyespot visual diagnoses did not correlate with the incidence of any of the pathogens assayed by PCR. Brown foot rot, caused principally by Microdochium nivale var . majus , accumulated earlier in the season than eyespot. Overall, visual diagnoses of stem base disease coincided poorly with PCR data until after growth stage (GS) 30.     

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.     

The escalating threat of Rhizoctonia cerealis, the causal agent of sharp eyespot in wheat

Rhizoctonia cerealis, the causal agent of sharp eyespot on wheat, was not considered to be an important pathogen for many years. Recently, the disease has become endemic in many countries except for South America. The disease has created a new threat to world wheat production because the damage of wheat sharp eyespot has become increasingly severe. In this paper, previous studies on this pathogen, including the disease geographical distribution, pathogen identification, life cycle, symptoms, favourable environmental conditions, effects on wheat yield and control strategy, are reviewed. Such information will be helpful in management of sharp eyespot.     

Screening for potential antagonists of Pseudocercosporella herpotrichoides, the causal agent of eyespot disease of cereals

Bacteria isolated from wheat seedlings, plants or straw from several field sites were screened for antagonism towards the cereal eyespot pathogen Pseudocercosporella herpotrichoides on several media of differing nutrient status. Thirteen out of 348 isolates inhibited pathogen growth on low-nutrient media and several also prevented spore germination or reduced germ tube extension. These were selected for further tests on wheat seedlings inoculated with the eyespot fungus. Twelve known bacterial antagonists of other fungal plant pathogens were tested in vitro using the same methods, and the majority showed some activity towards P. herpotrichoides. Selected isolates were equally inhibitory to both W and R pathotypes of the fungus. Effects of potential antagonists on disease development were assessed by scoring lesions or by counting the number of infection plaques formed by the fungus on leaf sheaths. Two isolates of Pseudomonas fluorescens , along with a commercial strain of Streptomyces griseoviridis , showed activity both in vitro and in subsequent infection trials with plants and may therefore be of potential value as antagonists of P. herpotrichoides.     

Influence of crop management on eyespot development and infection cycles of winter wheat

Wheat was assessed at four crop growth stages for eyespot (anamorph Pseudocercosporella herpotrichoides, teleomorph Tapesia yallundae) in a series of field trials that studied the effects on disease frequency of five wheat management techniques (sowing date and density, nitrogen fertiliser dose and form, removal/burial of cereal straw). An equation expressing disease level as a function of degree-days was fitted to the observed disease levels. This equation was based on eyespot epidemiology and depended on two parameters illustrating the importance of the primary and the secondary infection cycles respectively. Cultural practices were classified according to the importance of their effects on disease, and these effects could be related to infection cycles and host plant architecture. Sowing date had the earliest and strongest effect; early sowing always increased disease frequency through the primary infection cycle, and its influence on the secondary cycle was variable. Disease frequency was increased by high plant density and/or a low shoot number per plant through primary infection; the secondary cycle was, however, decreased by a low shoot number per plant, which reduced late disease development at high plant density. High nitrogen doses increased disease levels and the severity of both infection cycles, but this effect was partly hidden by a simultaneous stimulation of tillering and thus an indirect decrease of disease incidence. When significant, ammonium (vs ammonium nitrate) fertiliser decreased eyespot levels and infection cycles whereas straw treatment (burial vs removal of straw from the previous cereal crop) had no effect.     

Brachypodium distachyon exhibits compatible interactions with Oculimacula spp. and Ramularia collo‐cygni,providing the first pathosystem model to study eyespot and ramularia leaf spot diseases

Brachypodium distachyon (Bd) has established itself as an essential tool for comparative genomic studies in cereals and increasing attention is being paid to its potential as a model pathosystem. Eyespot and ramularia leaf spot (RLS) are important diseases of wheat, barley and other small‐grain cereals for which very little is known about the mechanisms of host resistance despite urgent requirements for plant breeders to develop resistant varieties. This work aimed to test the compatibility of interaction of two Bd accessions with the cereal pathogens Oculimacula spp. and Ramularia collo‐cygni, the causal agents of eyespot and RLS diseases, respectively. Results showed that both Bd accessions developed symptoms similar to those on the natural host for all pathogen species tested. Microscopy images demonstrated that R. collo‐cygni produced secondary conidia and both Oculimacula spp. formed characteristic infection structures on successive tissue layers. Visual disease assessment revealed that quantitative differences in disease severity exist between the two Bd accessions. The results presented here provide the first evidence that Bd is compatible with the main causal agents of eyespot and RLS diseases, and suggest that future functional genetic studies can be undertaken to investigate the mechanisms of eyespot and RLS disease resistance using Bd.     

Development of a Real-Time Polymerase Chain Reaction Assay for Quantifying Verticillium albo-atrum DNA in Resistant and Susceptible Alfalfa

ABSTRACT A precise real-time polymerase chain reaction (PCR) assay was developed for quantifying Verticillium albo-atrum DNA. The assay was used in a repeated experiment to examine the relationship between the quantity of pathogen DNA detected in infected leaves and shoots and the severity of Verticillium wilt symptoms in several alfalfa cultivars expressing a range of disease symptoms. Plants were visually inspected for symptoms and rated using a disease severity index ranging from 1 to 5, and the quantity of pathogen DNA present in leaves and stems was determined with real-time PCR. No significant differences in pathogen DNA quantity or disease severity index were observed for experiments or for cultivar-experiment interactions. Significant differences were observed between cultivars for the quantity of pathogen DNA detected with real-time PCR and also for disease severity index ratings. In both experiments, the highly resistant check cultivar Oneida VR had significantly less pathogen DNA, and significantly lower disease severity index ratings than the resistant cultivar Samauri, the moderately resistant cultivar Vernema, and the susceptible check cultivar Saranac. In both experiments, the Spearman rank correlation between the amount of V. albo-atrum DNA detected in leaves and stems with real-time PCR and disease severity index ratings based on visual examination of symptoms was positive (>0.52) and significant (P < 0.0001). These results suggest that resistance to Verticillium wilt in alfalfa is characterized by a reduced colonization of resistant genotypes by the fungus.     

Development of Oculimacula yallundae and O. acuformis (eyespot) lesions on stems of winter wheat in relation to thermal time in the UK

Relationships between development of eyespot, caused by Oculimacula yallundae (OY) or O. acuformis (OA) on stems of winter wheat (cv. Avalon), and thermal time (°C days after sowing) were investigated in field experiments in 1985/86, 1986/87 and 1987/88 (two experiments). In all experiments, the incidence and severity of stem eyespot (uninoculated plots, OY- and OA-inoculated plots) were linearly related to accumulated thermal time after sowing. There were ca. 600–800°C days from the time of the first sample when eyespot lesions were recorded on stems to the time when maximum eyespot incidence or severity was recorded. Relationships between stem eyespot incidence or severity and thermal time differed between seasons, with more severe eyespot in 1986/87 and 1987/88 than 1985/86. Both the severity and volume of stem lesions were initially greater in OY-inoculated plots than OA-inoculated plots in spring but differences were less by harvest in all seasons. The percentage of plants with stems colonized by OY or OA over all plots (including uninoculated) showed a consistent pattern in 1986/87 and 1987/88 (two experiments), with the percentage colonized by OY greater initially and the percentage colonized by OA gradually increasing with time towards harvest.     

Mining the Watkins collection of wheat landraces for novel sources of eyespot resistance

Eyespot is an economically important stem base disease of wheat caused by the soilborne fungal pathogens Oculimacula yallundae and Oculimacula acuformis. The most effective method of controlling the disease is host resistance. However, there are only three genetically characterized resistances in wheat varieties and further sources of resistance are required. Previous studies have identified resistances in wild relatives, but use of these resistances has been limited by linkage drag with deleterious traits exacerbated by low rates of recombination. Therefore, the identification of novel resistances in hexaploid wheat germplasm is desirable. The Watkins collection currently consists of 1056 hexaploid wheat landraces that represent global wheat diversity at the time of its collection in the 1920s and 1930s. As such, it may contain beneficial agronomic traits such as eyespot resistance. The Watkins collection was screened for resistance to O. yallundae based on a glasshouse test of all 1056 accessions and a polytunnel test of 44 accessions selected from a previous field trial. Resistant lines identified in these tests were retested against both O. yallundae and O. acuformis. This identified 17 accessions with resistance to one or both of the pathogen species. From these, two accessions (1190094.1 and 1190736.3) provided a high level of resistance to both pathogen species. An F₄ population derived from accession 1190736.3 indicated that the resistance to O. acuformis in this accession is conferred by a single gene and therefore would be suitable for introgression into elite wheat varieties to provide an alternative source of eyespot resistance.     

Development of specific PCR primers for diagnosis and quantitative detection of the fungal maize pathogen <Emphasis Type="Italic">Kabatiella zeae</Emphasis>

The fungus Kabatiella zeae which causes the eyespot disease in maize has become an important leaf disease in the northern regions of Europe. An early and accurate diagnosis is necessary for determining the first occurrence in the field. The visual assessment of this pathogen is often difficult because different spots, which may be parasitic, physiological or genetic complicate the exact diagnosis. The specific identification of the fungus is possible by PCR. Unfortunately, no specific DNA sequences are available in the databases. Hence, shot-gun cloning was done resulting in K. zeae-specific sequences for primer design. Cross testing with maize DNA and other pathogens, including Setosphaeria turcica, Mycosphaerella zeae-maydis and various Fusarium species, revealed that the primers were absolutely specific for K. zeae and can be used for qualitative and quantitative PCR assays.     

河北省小麦纹枯病菌群体组成及致病力分化

为明确河北省小麦纹枯病发生特点及病菌特征,采用五点取样法调查该病的发生情况,通过细胞核染色、菌丝融合反应和r DNA-ITS序列分析测定196株纹枯病菌群体组成,并比较其对不同小麦品种的致病力差异。结果表明,河北省3个不同生态类型麦区30个监测点均有纹枯病发生,临漳县发病最重,其次是邯郸县和馆陶县,青县发病最轻;纹枯病菌可划分为AG-D、AG-B(0)、AGI、AG-4和AG-5共5种融合群,分别占菌株总数的88.3%、1.5%、5.1%、3.6%和1.5%;采自黑龙港平原区和山前平原区的纹枯病菌对石新828、良星99和邯6172的平均致病力均明显强于冀东平原区;菌株可划分为极强、强、中等和弱4个致病类型,分别占菌株总数的45.92%、33.67%、7.65%和12.76%。表明河北省小麦纹枯病发生普遍,总体呈南重北轻的趋势,纹枯病菌群体组成较简单,以强致病力AG-D融合群为主。     

内生细菌EBS05在小麦体内的定殖动态及其对小麦纹枯病的防治作用

以抗药性突变菌株为筛选标记,采用稀释倒平板法,研究了内生细菌EBS05在小麦体内的定殖动态,并通过盆栽和大田试验研究了EBS05对小麦纹枯病的防治效果。结果表明,EBS05在小麦根内具有较强的定殖能力,且能从根部向茎、叶部转移,其定殖量与菌株的接种浓度呈正相关,当接种浓度为108 CFU/mL时,EBS05能在根、茎内有效定殖,并持续向叶内转移。以菌体浓度为108CFU/mL的发酵液进行浸种或灌根处理后,EBS05在根内定殖量始终大于同时期茎、叶内的定殖量;菌株在小麦根、茎和叶内的定殖数量均表现为接种初期逐渐增加,至接种后第10～12天达到最大值,随后逐渐降低。内生细菌EBS05对小麦纹枯病的防治效果优于对照药剂25 g/L咯菌腈（flu-dioxonil）悬浮种衣剂,带菌发酵液和除菌发酵液的盆栽防治效果分别达91.2%和88.2%,大田防治效果分别为66.3%和56.2%。     

The role of seeds and airborne inoculum in the initiation of leaf blotch (Rhynchosporium secalis) epidemics in winter barley

Both airborne spores of Rhynchosporium secalis and seed infection have been implied as major sources of primary inoculum for barley leaf blotch (scald) epidemics in fields without previous history of barley cropping. However, little is known about their relative importance in the onset of disease. Results from both quantitative real‐time PCR and visual assessments indicated that seed infection was the main source of inoculum in the field trial conducted in this study. Glasshouse studies established that the pathogen can be transmitted from infected seeds into roots, shoots and leaves without causing symptoms. Plants in the field trial remained symptomless for approximately four months before symptoms were observed in the crop. Covering the crop during part of the growing season was shown to prevent pathogen growth, despite the use of infected seed, indicating that changes in the physiological condition of the plant and/or environmental conditions may trigger disease development. However, once the disease appeared in the field it quickly became uniform throughout the cropping area. Only small amounts of R. secalis DNA were measured in 24 h spore‐trap tape samples using PCR. Inoculum levels equivalent to spore concentrations between 30 and 60 spores per m³ of air were only detected on three occasions during the growing season. The temporal pattern and level of detection of R. secalis DNA in spore tape samples indicated that airborne inoculum was limited and most likely represented rain‐splashed conidia rather than putative ascospores.     

Population dynamics of Rhizoctonia,Oculimacula, and Microdochium species in soil,roots, and stems of English wheat crops

This study aimed to elucidate the population dynamics of Rhizoctonia, Oculimacula, and Microdochium species, causing the stem base disease complex of sharp eyespot, eyespot, and brown foot rot in cereals. Pathogen DNA in soil, roots, and stem fractions, and disease expression were quantified in 102 English wheat fields in two seasons. Weather data for each site was collected to determine patterns that correlate with assessed diseases. Oculimacula spp. (66%) and R. solani AG 2-1 (63%) were most frequently detected in soil, followed by R. cerealis (54%) and Microdochium spp. (33%). Oculimacula spp. (89%) and R. cerealis (56%) predominated on roots and soil but were not associated with root rot symptoms, suggesting that these species used soil and roots for survival and as inoculum source. M. nivale was more frequently detected than M. majus on stems up to GS 21–30 and co-occurred on plant samples with O. acuformis. O. yallundae had higher DNA concentration than O. acuformis at the lower 5 cm basal region at GS 37–45. R. cerealis predominated in the upper 15 cm above the base beyond stem extension. Brown foot rot by Microdochium spp. was favoured by cool and wet autumns/winters and dominated in English wheat. Eyespot and sharp eyespot disease index by Oculimacula spp. and R. cerealis, respectively, correlated with wet/humid springs and summers. Results suggested that stem base pathogens generally coexisted; however, their abundance in time and space was influenced by favourable weather patterns and host development, with niche differentiation after stem extension.     

Differential seedling resistance to the eyespot pathogens,Oculimacula yallundae and Oculimacula acuformis,conferred by Pch2 in wheat and among accessions of Triticum monococcum

Eyespot is an economically important stem‐base disease of wheat caused by two fungal species: Oculimacula yallundae and Oculimacula acuformis. This study investigated the efficacy of two sources of resistance, viz. the genes Pch1, introgressed into hexaploid wheat from Aegilops ventricosa, and Pch2, identified in wheat cv. Cappelle Desprez, against O. yallundae and O. acuformis separately. In a series of seedling bioassays Pch1 was found to be highly effective against both species. Although Pch2 was found to confer resistance against both pathogen species, it was significantly less effective against penetration from O. yallundae than O. acuformis. Furthermore, a quantitative trait locus (QTL) analysis was not able to locate any resistance to O. yallundae on chromosome 7A of Cappelle Desprez. This has important implications for the use of Pch2 in commercial cultivars as it is necessary to have genes that confer resistance to both pathogens for effective eyespot control. In addition, a set of 22 T. monococcum accessions was screened for resistance to both O. yallundae and O. acuformis to identify potentially novel resistances and to assess the accessions for evidence of differential resistance to the eyespot species. Significant differences in resistance to the two pathogens were identified in four of these lines, providing evidence for differential resistance in T. monococcum. This study demonstrates that future screening for novel sources of eyespot resistance should investigate both pathogen species.     

Pseudocercosporella anguioides, a weakly pathogenic fungus associated with eyespot in winter wheat at a site in England

Pseudocercosporella anguioides is reported for the first time from Britain. Isolates were made from the leaf sheaths of four plants of a sample of 2716 with suspected eyespot lesions taken in April 1986 from a winter wheat crop. The eyespot pathogen, P. herpotrichoides , was isolated from 724 plants in the same sample. P. anguioides was not found in a sample of eyespot-infected straws taken from the same crop in July 1986. In infection tests, the P. anguioides isolates produced no obvious lesions on the leaf sheaths of wheat seedlings grown in pots, but were sometimes reisolated from symptomless leaf sheaths. Although P. anguioides occurred infrequently, care is needed to distinguish it from P. herpotrichoides when monitoring the eyespot pathogen.     

Evaluation of fungicides for control of eyespot disease and yield loss nationships in winter wheat

Following the discovery of resistance to benzimidazole fungicides in the cereal eyespot pathogen Pseudocercosporella herpotrichoides in the UK in 1981, and an initial in vitro screen to select the fungicides with greatest activity against the pathogen, 40 field experiments were carried out between 1983 and 1986 to evaluate alternative fungicides for control of eyespot. At the majority of experimental sites, benomyl-resistant strains of the pathogen were present, and carbendazim did not control eyespot. Prochloraz was the most effective fungicide, reducing the eyespot index by 30–60%. There was no extra benefit from adding carbendazim to prochloraz. Flusilazole was almost as effective as prochloraz, but other fungicides had little or no effect. At sites with a high incidence of eyespot, prochloraz, with or without the addition of carbendazim, generally gave the largest yield increase. The mean yield increases each year were in the range 0·36–0·85 t/ha, and the greatest yield increase at any site was 2·27 t/ha. Most other fungicides increased yield, but carbendazim did not from 1984 to 1986. There were also yield increases at many sites with a low incidence of eyespot. Yield increases were associated with increases in thousand-grain weight at the majority of sites, but in only a few instances were there associated increases in specific weight. Prochloraz application at GS30-31 was cost effective at 71% of sites. At most sites, in regression of yield on eyespot, eyespot accounted for less than 25% of the variance in yield. The mean relationship between severe eyespot lesions and yield loss was such that each 1% increase in the percentage of tillers affected by severe eyespot was associated with a yield loss of 0·21 %. There was a significant positive correlation between eyespot at GS75 and GS30-31, and between yield increase from prochloraz treatment (at GS30-31) and eyespot at GS75, but not between yield increase and eyespot at GS30-31. The ADAS threshold for fungicide application of 20% tillers affected at GS30-31 was a reliable indicator of the cost-effectiveness of treatment at 60% of sites.     

Disease severity and yield of pure-line wheat cultivars and mixtures in the presence of eyespot, yellow rust, and their combination

Five winter wheat cultivars, six two-component cultivar mixtures, and one four-way mixture were grown in the presence of yellow rust, eyespot, both diseases, and neither disease for three seasons. On average, mixtures reduced the severity of yellow rust relative to their component pure stands by 53%. The four-component mixture provided better yellow rust control than did the two-way mixtures. Eyespot severity was reduced through mixing only in the absence of yellow rust and by only three of the seven mixtures (mean reduction = 13%). Yellow rust was 13% less severe in the presence of eyespot, and eyespot was 10% more severe in the presence of yellow rust. Averaged over all years, the mixtures increased yield relative to the pure stands by 6·2, 1·7, 7·1, and 1·3% in the presence of yellow rust, eyespot, both diseases, and neither disease, respectively. Two mixtures provided significant yield increases over the means of their component pure stands (7% and 9%) in the presence of eyespot even though one of them did not significantly reduce eyespot severity. Accounting for all disease treatments and years, four mixtures provided distinctly higher yield increases than the other three. In mixtures containing a resistant cultivar and a cultivar susceptible to eyespot, yield loss by the susceptible cultivar was not compensated for by increased yield of the resistant cultivar. The mixtures showed improved yield stability relative to the pure stands, with the four-component mixture being particularly stable.