Wheat Sharp Eyespot Research Articles

Development of a Rapid Approach for Detecting Sharp Eyespot Resistance in Seedling-Stage Wheat and Its Application in Chinese Wheat Cultivars.

Sharp eyespot, caused by Rhizoctonia cerealis, has become one of the most severe diseases affecting global wheat production in recent decades. Quick and efficient screening methods are required to accelerate the development of cultivars for sharp eyespot resistance in wheat breeding. Here, a two-step colonized wheat kernels (TSCWK) method for the inoculation and classification of sharp eyespot resistance in seedlings was established in a greenhouse. After preliminary verification of the reliability of the method in two replicates, 196 wheat cultivars were assessed for sharp eyespot resistance, and significant correlations were identified among the four replicates (r = 0.78 to 0.84; P < 0.01). Furthermore, the 196 cultivars were scored for sharp eyespot resistance at the milk-ripe stage using traditional toothpick inoculation in the field. Correlation and linear regression analysis showed that the application of this approach at the seedling stage showed good consistency with the traditional field method. Moreover, the scoring of 442 cultivars using the TSCWK method indicated that most cultivars from the Huanghuai valley were susceptible to R. cerealis, suggesting an urgent need to improve sharp eyespot resistance in this region. Additionally, the relative resistance index of sharp eyespot decreased in the surveyed cultivars of the region with time. This study offers a rapid and effective approach for the identification of wheat sharp eyespot resistance and provides valuable germplasm for improving sharp eyespot resistance in wheat breeding.

Use of resistant Rhizoctonia cerealis strains to control wheat sharp eyespot using organically developed pig manure fertilizer

The effects of Rhizoctonia cerealis resistant strains to control wheat sharp eyespot in pig manure developed organic fertilizer were investigated. Organic fertilizer was produced in a self-built aerated static composting box (0.37 m3) and later assessed using values of temperature, moisture, pH, C/N ratio, nitrogen transformation, fluvic- and humic acid composition and germination index. Together, these assessments indicated that disposed pig manure could be considered as a non-hazardous fertilizer. By adding R. cerealis resistant strains (Bacillus flexus, B. amyloliquefaciens, B. amyloliquefaciens, B. licheniformis and Paenibacillus sp.), we were successfully able to develop wheat sharp eyespot inhibiting bio-organic fertilizer. Our results showed that soil applied with 10 t/hm2 of the developed bio-organic fertilizer significantly improved the dry weight of the wheat, promoted chlorophylls and soluble protein composition and effectively prevented the wheat from sharp eyespot. The protection rate (77.1%) of our developed bio-organic fertilizer is 42.3% higher than the tested commercially available organic fertilizer (34.8%), and the disease index is significantly lower (P < 0.001) than both the control and commercial organic fertilizer. In conclusion, the prevention and control effects of our developed bio-organic fertilizer on wheat sharp eyespot were both significant and promising.

Characterization of a Versatile Plant Growth-Promoting Rhizobacterium Pseudomonas mediterranea Strain S58.

Plant growth-promoting rhizobacterial strain S58 was isolated from the tobacco rhizosphere. It showed strong antagonism against a battery of plant pathogenic fungi and bacteria, and controlled wheat sharp eyespot and tobacco wildfire diseases efficiently. Further tests showed that strain S58 solubilized organic phosphate and produced siderophore, protease, ammonia, and indole-3-acetic acid. In Arabidopsis thaliana, it promoted plant growth and changed root system architecture by restricting the growth of primary roots and increasing lateral root numbers. We relied on morphological, biochemical, physiological characteristics, and molecular phylogenic analysis to identify strain S58 as Pseudomonas mediterranea. The complete genome of strain S58 has a single circular chromosome of 6,150,838 bp with a 61.06% G+C content. The bacterial genome contained 5,312 predicted genes with an average length of 992.90 bp. A genome analysis suggested that P. mediterranea S58 was a rich cyclic lipopeptide (CLP)-producing strain that possessed seven non-ribosomal peptide gene clusters for CLP synthesis. Leaf inoculation of the bacterial culture and supernatants triggered cell death-like immunity in tobacco. Quantitative real-time PCR assays showed that the strain S58 induced the expression of pattern-triggered immunity and cell death marker genes, but not jasmonic acid marker genes. The results suggested that P. mediterranea S58 is a novel, versatile plant growth-promoting agent with multiple beneficial traits for plants.

Ecotopic Expression of the Antimicrobial Peptide DmAMP1W Improves Resistance of Transgenic Wheat to Two Diseases: Sharp Eyespot and Common Root Rot.

Wheat (Triticum aestivum L.) is an important staple crop. Sharp eyespot and common root rot are destructive diseases of wheat. Antimicrobial peptides (AMPs) are small peptides with broad-spectrum antimicrobial activity. In this study, we synthesized the DmAMP1W gene, encoding Dahlia merckii DmAMP1, and investigated the antifungal role of DmAMP1W in vitro and in transgenic wheat. Protein electrophoresis analysis and in vitro inhibition results demonstrated that the synthesized DmAMP1W correctly translated to the expected peptide DmAMP1W, and the purified peptide inhibited growths of the fungi Rhizoctonia cerealis and Bipolaris sorokiniana, the pathogenic causes of wheat sharp eyespot and common root rot. DmAMP1W was introduced into a wheat variety Zhoumai18 via Agrobacterium-mediated transformation. The molecular characteristics indicated that DmAMP1W could be heritable and expressed in five transgenic wheat lines in T1–T2 generations. Average sharp eyespot infection types of these five DmAMP1W transgenic wheat lines in T1–T2 generations decreased 0.69–1.54 and 0.40–0.82 compared with non-transformed Zhoumai18, respectively. Average common root rot infection types of these transgenic lines and non-transformed Zhoumai18 were 1.23–1.48 and 2.27, respectively. These results indicated that DmAMP1W-expressing transgenic wheat lines displayed enhanced-resistance to both sharp eyespot and common root rot. This study provides new broad-spectrum antifungal resources for wheat breeding.

Genetic structure and population diversity in the wheat sharp eyespot pathogen Rhizoctonia cerealis in the Willamette Valley, Oregon, USA

Wheat sharp eyespot, caused by the soilborne basidiomycete fungus Rhizoctonia cerealis group D subgroup I (AG‐DI), is a common disease of wheat stems that has increased in global importance in recent years. The disease caused a severe and extensive epidemic throughout the Willamette Valley of Oregon in 2014 and has remained one of the most important wheat diseases in this region subsequently. The population structure of this pathogen is still not well understood. In this study, 572 pure cultures of R. cerealis AG‐DI were isolated from five commercial fields and two experimental fields in the Willamette Valley, Oregon, in the spring of 2017 and 2018. Population structure studies were conducted using six microsatellite markers. Polymorphisms were observed at all six loci, and all seven subpopulations showed moderate gene and genotype diversity. Most of the loci were in Hardy–Weinberg equilibrium in the subpopulations, the entire population combined over locations and five of seven subpopulations were in gametic equilibrium, and the AMOVA and Mantel’s test demonstrated no differentiation among subpopulations. Though direct evidence is currently lacking, these results suggest that the sexual stage of the pathogen may be having a significant impact on pathogen population structure, and that the presence of basidiospores could be contributing to the extensive spread of this disease in the Willamette Valley in recent years.

Development and Application of Seed Coating Agent for the Control of Major Soil-Borne Diseases Infecting Wheat

In order to reduce the usage amount of pesticide fertilizers and protect the natural environment, seed coating agents are receiving increased wide concern. In this study, the active constituent (pesticide) and inactive components (surfactants and film former) of the seed coating agents were screened and optimized by the wet sand processing superfine grinding method. The fungal inhibition test of pesticides showed that thifluzamide, fludioxonil, pyraclostrobin, and difenoconazole have an obvious fungal inhibitory effect on wheat sharp eyespot, take-all, and root rot. LAE-9 and polyacrylamide + carboxymethyl cellulose (CMC) is recommended for the safe surfactant and film former, respectively, based on the seed germination test. Moreover, 6% difenoconazole · fludioxonil flowable concentrate for seed coating (FSC) stimulates the seedling growth of wheat, advances the growth of root, and improves biomass in the field trial, meanwhile, the control efficiency reached above 80%. Thus, we suggested it can be used as an effective seed coating agent for the control of soil-borne diseases in wheat. The seed coating agent has the characteristics of disease prevention, increasing crop yield, and safety of environment, which is of significance in practical application.

Development and molecular cytogenetic identification of a new wheat-rye 4R chromosome disomic addition line with resistances to powdery mildew, stripe rust and sharp eyespot.

A wheat-rye 4R chromosome disomic addition line with resistances to powdery mildew, stripe rust, sharp eyespot and high kernel number per spike was developed and characterized by molecular cytogenetic method as novel resistant germplasm. Rye (Secale cereale L.), a close relative of common wheat, is an important and valuable gene donor with multiple disease resistance for wheat improvement. However, resistance genes derived from rye have successively lost resistance to pathogens due to the coevolution of pathogen virulence and host resistance. Development and identification of new effective resistance gene sources from rye therefore are of special importance and urgency. In the present study, a wheat-rye line WR35 was produced through distant hybridization, embryo rescue culture, chromosome doubling and backcrossing. WR35 was then proven to be a new wheat-rye 4R disomic addition line using sequential GISH (genomic in situ hybridization), mc-FISH (multicolor fluorescence in situ hybridization) and ND-FISH (non-denaturing FISH) with multiple probes, mc-GISH (multicolor GISH), rye chromosome arm-specific marker analysis and SLAF-seq (specific-locus amplified fragmentsequencing) analysis. At the adult stage, WR35 exhibited high levels of resistance to the powdery mildew (Blumeria graminis f. sp. tritici, Bgt) and stripe rust (Puccinia striiformis f. sp. tritici, Pst) pathogens prevalent in China, and a highly virulent isolate of Rhizoctonia cerealis, the cause of wheat sharp eyespot. At the seedling stage, it was highly resistant to 22 of 23 Bgt isolates and four Pst races. Based on its disease responses to different pathogen isolates, WR35 may possess resistance gene(s) for powdery mildew, stripe rust and sharp eyespot, which differed from the known resistance genes from rye. In addition, WR35 was cytologically stable and produced high kernel number per spike. Therefore, WR35 with multi-disease resistances and desirable agronomic traits should serve as a promising bridging parent for wheat chromosome engineering breeding.

Use of Lentinan To Control Sharp Eyespot of Wheat, and the Mechanism Involved.

Lentinan (LNT), a complex polysaccharide with a β-(1→3)-linked backbone of d-glucose residues, has been reported to inhibit plant diseases. Our objective was to explore the efficacy and action mechanism of LNT used as a seed dressing to control sharp eyespot of wheat. Seed dressing promoted wheat growth. At control germination rates of 50%, 8 g of LNT/100 kg of seeds of the Jimai 22, Shannong 23, and Luyuan 502 cultivars significantly increased seed germination to 54%, 52%, and 51%, respectively. Seven days after emergence, the heights and root activity of wheat treated with LNT were significantly greater than those of controls. These effects were dose-dependent. At this time, the plant heights of Jimai 22, Shannong 23, and Luyuan 502 cultivars were 9.52, 8.52, and 10.52 cm, respectively, significantly higher than that of the controls. LNT prevented the development of wheat sharp eyespot. In the highly susceptible Jimai 22 cultivar, sharp eyespot development was reduced by 33.7%, 31.9%, and 30.4% at 7, 14, and 21 days after germination. LNT somewhat increased phenylalanine ammonia-lyase, peroxidase, and superoxide dismutase activity; reduced the malondialdehyde content; increased chlorophyll a and b levels; and enhanced the root vigor of wheat. These effects peaked 7 days after germination. LNT increased transcription of the genes encoding alternative oxidase (AOX) and β-1,3-glucanase (GLU), the salicylic acid signaling pathway-related gene NbPR1a, and the sharp eyespot resistance-related gene RS33. A significant dose-effect relationship was evident in terms of AOX transcription; we thus speculate that AOX may be the target gene.

Genetic Structure of Populations of the Wheat Sharp Eyespot Pathogen Rhizoctonia cerealis Anastomosis Group D Subgroup I in China.

Sharp eyespot on wheat is caused by Rhizoctonia cerealis anastomosis group D subgroup I (AG-DI) and is an economically important stem-base disease of wheat in temperate regions worldwide. However, the understanding about the field population structure of R. cerealis is limited. In this study, the genetic structure of four wheat-infecting populations in China was investigated using six microsatellite markers characterized from the transcriptome data of R. cerealis AG-DI. A total of 173 unique genotypes were identified among 235 fungal isolates. Departure from Hardy-Weinberg equilibrium, a significant degree of inbreeding, and a significant deficit in heterozygotes indicated a nonrandom mating pattern. Combining the low to intermediate degrees of gametic disequilibrium, although with high genotypic diversity and low to moderate clonal fractions, sexual reproduction probably existed, but the asexual reproduction should be the predominant reproductive mode. Structural analysis showed three gene pools among the four populations, which indicated the existence of three evolutionary origins of R. cerealis AG-DI. The long-distance movement of contaminated material, especially the infected seed, might have caused the moderate gene flow among these populations, which was consistent with the high differentiation among these populations. Overall, the genetic characteristics of the populations suggested a moderate evolutionary potential for R. cerealis AG-DI in China.

Homozygous and heterozygous point mutations in succinate dehydrogenase subunits b, c and d of Rhizoctonia cerealis conferring resistance to thifluzamide.

Thifluzamide, a succinate dehydrogenase inhibitor (SDHI) fungicide, is a promising fungicide for controlling wheat sharp eyespot (WSE). WSE is caused by Rhizoctonia cerealis. Information on the resistance mechanism of this pathogen to thifluzamide remains unavailable. We used selective reculturing and UV mutagenesis to generate thifluzamide-resistant mutants. Thifluzamide-resistant mutants were only generated through UV mutagenesis. Sequence analysis of succinate dehydrogenase (Sdh) genes revealed that two mutants had no mutation in RCSdhB, RCSdhC and RCSdhD, and the other 18 mutants all had at least one mutation in RCSdhB, RCSdhC or RCSdhD, either in a homozygous or heterozygous state. The majority of mutants included either RCSdhD-H116Y or RCSdhC-H139Y. They showed slight resistance to boscalid, bixafen and penflufen. Only one mutant possessed RCSdhB-H246Y, and it showed medium resistance to boscalid and penflufen and a slight resistance to bixafen. All the thifluzamide mutants were sensitive to flutolanil. Compared with their parental isolates, these mutants present no or minor fitness penalties. Homozygous and heterozygous point mutations in the succinate dehydrogenase subunits b, c and d of R. cerealis may be involved in thifluzamide resistance. © 2016 Society of Chemical Industry.

Characterization of Rhizoctonia cerealis sensitivity to thifluzamide in China

Wheat sharp eyespot (WSE) caused by Rhizoctonia cerealis is a major soilborne fungal pathogen in winter wheat in China. The succinate dehydrogenase inhibitor (SDHI) fungicide thifluzamide has significant fungicidal activity against Rhizoctonia spp., but the sensitivity of R. cerealis and the efficacy of thifluzamide for the control of R. cerealis are unknown. One-hundred sixty-seven R. cerealis isolates were collected from Jiangsu Province in China, and their sensitivity to thifluzamide was determined in vitro. Of these, seventy-seven isolates collected in 1984 and 2001 from 53 fields with no previous history of exposure to SDHI fungicides were used to establish baseline sensitivity. Baseline EC50 (effective concentration that results in 50% of mycelial growth inhibition) values for thifluzamide ranged from 0.0145 to 0.0764 (0.0370 ± 0.0014) mg l−1. The EC50 values for thifluzamide inhibition in the other 90 isolates collected in 2008, 2010 and 2013 from 62 fields with a history of likely exposure to SDHI fungicides, ranged from 0.0178 to 0.0889 (0.0382 ± 0.0015) mg l−1. The mean EC50 value of the baseline isolates was not significanty different than the mean EC50 value of the isolates collected after the introduction of the fungicide. In field trials in 2013 and 2014, the efficacy of thifluzamide applied at the late tillering stage [Zadok's growthstage (GS) 29)] at a concentration of 72.0 g a.i. ha−1 ranged from 75.8% to 91.7%. The efficacy of thifluzamide applied at the early stem elongation stage (GS 30) at a concentration of 72.0 g a.i. ha−1 ranged from 77.9% to 86.3%. The efficacy of jinggangmycin, a fungicide extensively used in China, ranged from 34.3% to 73.1% during 2012–2014. These results suggest that thifluzamide is very effective against WSE and could be a good alternative fungicide to jinggangmycin.

The phosphotransferase system gene ptsI in the endophytic bacterium Bacillus cereus is required for biofilm formation, colonization, and biocontrol against wheat sharp eyespot.

Natural resistance of wheat plants to wheat sharp eyespot is inadequate, and new strategies for controlling the disease are required. Biological control is an alternative and attractive way of reducing the use of chemicals in agriculture. In this study, we investigated the biocontrol properties of endophytic bacterium Bacillus cereus strain 0-9, which was isolated from the root systems of healthy wheat varieties. The phosphotransferase system is a major regulator of carbohydrate metabolism in bacteria. Enzyme I is one of the protein components of this system. Specific disruption and complementation of the enzyme I-coding gene ptsI from B.cereus was achieved through homologous recombination. Disruption of ptsI in B.cereus caused a 70% reduction in biofilm formation, a 30.4% decrease in biocontrol efficacy, and a 1000-fold reduction in colonization. The growth of ΔptsI mutant strain on G-tris synthetic medium containing glucose as the exclusive carbon source was also reduced. Wild-type properties could be restored to the ΔptsI mutant strain by ptsI complementation. These results suggested that ptsI may be one of the key genes involved in biofilm formation, colonization, and biocontrol of B.cereus and that B.cereus wild-type strain 0-9 may be an ideal biocontrol agent for controlling wheat sharp eyespot.

Metabolism of fungicide 2-allylphenol in Rhizoctonia cerealis

2-Allylphenol is a biomimetic synthetic fungicide that mimics the compound ginkgol found in gingko fruit (Gingko biloba L.). This systemic fungicide can effectively suppress a wide range of plant diseases, including wheat sharp eyespot (Rhizoctonia cerealis). However, its degradation in environment after application is still unknown. To understand this fungicide degradation, major metabolites of 2-allylphenol in R. cerealis were examined. The parent and metabolites of 2-allylphenol were detected and quantified in the mycelia and liquid medium. Results showed that 2-allylphenol was metabolized and bio-transformed by R. cerealis, and four metabolites were found, including 2-(2-hydroxyphenyl) acetic acid (M1), 2-(2, 3-dihydroxypropyl) phenol (M2), 2-(2-hydroxypropyl)-phenol (M3) and 2-(3-hydroxypropyl)-phenol (M4). Based on the results, we propose that the biodegradation pathway is that 2-allylphenol is rapidly oxidized into metabolite M2 and hydrolyzed into M3 and M4, which formed M2, and carboxylation of M2 to 2-hydroxy-3-(2׳-hydroxyphenyl) propionic acid which undergo hydrolyzation and decarboxylation to form M1. 2-Allylphenol can be bio-transformed to new compounds by R. cerealis, suggesting the existence of microbe metabolic pathways for 2-allylphenol.

Combined application of Bacillus subtilis NJ-18 with fungicides for control of sharp eyespot of wheat

Antagonistic bacterium, Bacillus subtilis NJ-18, can biologically control several plant diseases. A combined strategy of wheat sharp eyespot control consisting of B. subtilis NJ-18 and fungicides was evaluated. Greenhouse and field tests were conducted to determine the effect of combining the B. subtilis NJ-18 with the fungicides flutolanil and difenoconazole for the control of wheat sharp eyespot caused by Rhizoctonia cerealis Van der Hoeven. The sensitivity of B. subtilis NJ-18 to fungicides flutolanil and difenoconazole was tested. The growth of NJ-18 was unaffected by flutolanil in a broth medium, and the survival of NJ-18 spores on wheat seed was unaffected by difenoconazole. In greenhouse experiments, disease control obtained with a combination of NJ-18 and either fungicide was better than the control obtained with the bacterium or fungicides alone, and some combinations of bacterium plus fungicide demonstrated a small synergistic effect in reducing disease. Similar results were obtained with repeated field experiments. In addition to providing a highly effective disease control, the use of combinations enabled a reduced level of fungicide application. Overall, the results suggest that the combination of B. subtilis strain NJ-18 and flutolanil or difenoconazole represents a promising new tool for the control of sharp eyespot of wheat.

Complete genome sequence of a novel endornavirus in the wheat sharp eyespot pathogen Rhizoctonia cerealis

We report here the presence of a novel double-stranded RNA (dsRNA) virus in an isolate (R0959) of the fungus Rhizoctonia cerealis, the causal agent of sharp eyespot of wheat in China. Sequence analysis showed that the dsRNA segment is 17,486 bp long and contains a single open reading frame (ORF) with the potential to encode a protein of 5,747 amino acids. The predicted protein contains conserved motifs of putative viral methyltransferase, helicase 1, and RNA-dependent RNA polymerase. Sequence similarity and phylogenetic analysis clearly place it in a distinct species within the genus Endornavirus, family Endornaviridae, and therefore we propose its name to Rhizoctonia cerealis endornavirus 1 (RcEV1). This is the first report of the full-length genomic sequence of a dsRNA mycovirus in R. cerealis.

Characterisation of Pseudomonas chlororaphis subsp. aurantiaca strain Pa40 with the ability to control wheat sharp eyespot disease

AbstractThis study details the isolation and characterisation of Pseudomonas chlororaphis subsp. aurantiaca strain Pa40, and is the first to examine P. chlororaphis for use in suppression of wheat sharp eyespot on wheat. Pa40 was isolated during an investigation aimed to identify biocontrol agents for Rhizoctonia cerealis. Over 500 bacterial strains were isolated from the rhizosphere of infected wheat and screened for in vitro antibiosis towards R. cerealis and ability to provide biocontrol in planta. Twenty‐six isolates showed highly antagonistic activity towards R. cerealis, in which Pseudomonas spp. and Bacillus spp. were predominant members of the antagonistic community. Strain Pa40 exhibited clear and consistent suppression of wheat sharp eyespot disease in a greenhouse study and suppression was comparable to that of chemical treatment with validamycin A. Pa40 was identified as P. chlororaphis subsp. aurantiaca by the Biolog identification system combined with 16S rDNA, atpD, carA and recA sequence analysis and biochemical and physiological characteristics. To determine broad‐spectrum applicability and the specific mechanisms involved in Pa40's pathogen suppression this strain was tested for antibiosis towards various phytopathogens and assayed for many biocontrol activities and plant‐beneficial traits. Strain Pa40 inhibited the growth of 10 of 13 phytopathogenic fungal strains and six of eight phytopathogenic bacteria tested. This original work characterises HCN, protease and siderophore production in P. chlororaphis. Each of these characteristics likely contributed to Pa40's biocontrol capabilities as well as stimulation of the hypersensitive response in tobacco and the presence of genes involved in the biosynthesis of phenazine, 2‐hydroxylated phenazine and pyrrolnitrin.

The heterogeneity of the rDNA-ITS sequence and its phylogeny in Rhizoctonia cerealis, the cause of sharp eyespot in wheat

The sequence heterogeneity of the ribosomal internal transcribed spacer (ITS) region was investigated for Rhizoctonia cerealis isolates from the anastomosis group AG-DI. Although sequence variability of the ITS has been reported in a few multinucleate R. solani isolates, it has very rarely been reported in binucleate Rhizoctonia spp. isolates and has never been described in R. cerealis, the pathogen of wheat sharp eyespot. In this study, the ITS regions of 15 R. cerealis isolates were cloned and sequenced. The results revealed more than one different ITS sequence within each isolate. This is the first evidence of ITS sequence heterogeneity in R. cerealis. Based on these ITS sequences, different sequences of one isolate did not cluster in one clade, but all of the sequences of the 15 isolates were clustered in the anastomosis subgroup AG-DI, suggesting that the heterogeneity of the ITS did not affect the molecular identification of their anastomosis group. Haplotype analyses indicated that there might be three evolutionary origins of R. cerealis, or a recombination event could be the cause of different ITS sequences in one genome. This study demonstrates the variability and the evolution of Rhizoctonia, especially binucleate R. cerealis. These findings will help design disease control strategies.

Purification and Structural Analysis of Surfactin Produced by Endophytic Bacillus subtilis EBS05 and its Antagonistic Activity Against Rhizoctonia cerealis

Bacillus subtilis EBS05, an endophytic bacteria strain isolated from a medicinal plant Cinnamomum camphor, can produce antagonistic compounds that effectively inhibit plant pathogenic fungi. The greenhouse experiments showed that wheat sharp eyespot disease (WSED) was reduced by 91.2%, 88.2% and 43.0% after the treatment with fermentation broth, bacteria-free filter and a fungicide fludioxonil, respectively. The culture broth of strain EBS05 can more effectively control WSED than can fludioxonil. The fermentation broth and bacteria-free filter ability to suppress WSED was not significantly different, suggesting that an active secreted substance played a major role in controlling WSED. Separation and purification of the active compounds was carried out by serial processes, including hydrochloric acid (pH 2.0) treatment, methanol extraction and Sephadex LH-20 column chromatography, silica gel column chromatography and reverse-phase high-pressure liquid chromatography (HPLC), respectively. The purified compounds, one of active peaks in the HPLC spectrum, were obtained from the collection. Analysis of the chemical structures by time-of-flight mass spectrometry (TOF-MS) and electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS) showed that the active substances produced by the endophytic bacteria EBS05 are mixture of the <TEX>${\beta}$</TEX>-hydroxy-C12~C15-<TEX>$Leu^7$</TEX> surfactin A isomers with 1035.65 Da, 1021.64 Da, 1007.63 Da and 993.65 Da molecular weights, respectively.

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.

Sensitivity to iprodione, difenoconazole and fludioxonil of Rhizoctonia cerealis isolates collected from wheat in China

Since the 1980s wheat sharp eyespot (WSE) caused mainly by Rhizoctonia cerealis has become one of serious diseases of wheat in China. In this study, the sensitivity of 89 R. cerealis isolates to different fungicides was evaluated using mycelial growth inhibition assays. The results showed that all R. cerealis isolates tested were sensitive to iprodione, difenoconazole and fludioxonil with mean EC 50 (effective concentration that results in 50% of mycelial growth inhibition) values of 0.419, 0.062 and 0.033 μg/ml, respectively. To evaluate the risk of fungicide resistance in R. cerealis, an attempt was made to induce resistant mutants in the laboratory. Although difenoconazole- and fludioxonil-resistant mutants were not obtained, we obtained seven independently iprodione-resistant (IR) mutants from 89 parental isolates. The EC 50 values for these IR mutants were greater than 100 μg/ml, whereas those for the original wild-type counterparts were less than 1 μg/ml. After having been subcultured on PDA for 10 generations, the IR mutants did not show any decrease in resistance to iprodione. Additionally, these IR mutants also showed resistance to fludioxonil but remained sensitive to difenoconazole. Osmotic sensitivity tests showed that the IR mutants were hypersensitive to osmotic stress generated by NaCl. Inoculation tests showed that all the IR mutants lost their ability to infect the host plant. Taken together, these results indicate that the current population of R. cerealis is sensitive to these fungicides and a fitness cost is associated with iprodione-resistant mutants of R. cerealis in both osmotic stress and pathogenicity. The information obtained in this study is useful in monitoring and managing fungicide resistance in R. cerealis populations in China.