Cereal Isolates Research Articles

Biodiversity of Lactobacillus helveticus isolates from dairy and cereal fermentations reveals habitat-adapted biotypes.

For the present study, we collected 22 Lactobacillus helveticus strains from different dairy (n=10) and cereal (n=12) fermentations to investigate their biodiversity and to uncover habitat-specific traits. Biodiversity was assessed by comparison of genetic fingerprints, low-molecular-weight subproteomes, metabolic and enzymatic activities, growth characteristics and acidification kinetics in food matrices. A clear distinction between the dairy and cereal strains was observed in almost all examined features suggesting that the different habitats are domiciled by different L. helveticus biotypes that are adapted to the specific environmental conditions. Analysis of the low-molecular-weight subproteome divided the cereal isolates into two clusters, while the dairy isolates formed a separate homogeneous cluster. Differences regarding carbohydrate utilization were observed for lactose, galactose, sucrose and cellobiose as well as for plant-derived glucosides. Enzymatic differences were observed mainly for ß-galactosidase and ß-glucosidase activities. Further, growth temperature was optimal in the range from 33 to 37°C for the cereal strains, whereas the dairy strains showed optimal growth at 40°C. Taken together, adaptation of the various biotypes results in a growth benefit in the particular environment. Acidification and growth tests using either sterile skim milk or a wheat flour extract confirmed these results. Differentiation of these biotypes and their physiological characteristics enables knowledge-based starter culture development for cereal versus dairy products within one species.

Characterization of Nivalenol-Producing Fusarium asiaticum That Causes Cereal Head Blight in Korea.

Fusarium asiaticum of the F. graminearum species complex causes head blight in small-grain cereals. The nivalenol (NIV) chemotypes of F. asiaticum is more common than the deoxynivalenol (DON) chemotypes of F. asiaticum or F. graminearum in Korea. To understand the prevalence of F. asiaticum–NIV in Korean cereals, we characterized the biological traits of 80 cereal isolates of F. asiaticum producing NIV or 3-acetyl-deoxynivalenol (3-ADON), and 54 F. graminearum with 3-ADON or 15-acetyl-deoxynivalenol (15-ADON). There was no significant difference in mycelial growth between the chemotypes, but F. asiaticum isolates grew approximately 30% faster than F. graminearum isolates on potato dextrose agar. Sexual and asexual reproduction capacities differed markedly between the two species. Both chemotypes of F. graminearum (3-ADON and 15-ADON) produced significantly higher numbers of perithecia and conidia than F. asiaticum–NIV. The highest level of mycotoxins (sum of trichothecenes and zearalenone) was produced by F. graminearum–3-ADON on rice medium, followed by F. graminearum–15-ADON, F. asiaticum–3-ADON, and F. asiaticum–NIV. Zearalenone levels were correlated with DON levels in some chemotypes, but not with NIV levels. Disease assessment on barley, maize, rice, and wheat revealed that both F. asiaticum and F. graminearum isolates were virulent toward all crops tested. However, there is a tendency that virulence levels of F. asiaticum–NIV isolates on rice were higher than those of F. graminearum isolates. Taken together, the phenotypic traits found among the Korean F. asiaticum–NIV isolates suggest an association with their host adaptation to certain environments in Korea.

Pathogenicity and Fungicide Sensitivity of Rhizoctonia solani and R. cerealis Isolates

The Rhizoctonia solani species consists of multinucleate isolates that belong to anastomosis groups AG1–AG3 and differ in virulence and host affinity. R. cerealis is a binucleate species of anastomosis group AG-D which causes sharp eyespot, a common plant disease in Poland. Rhizoctonia spp. is a ubiquitous soil pathogen that poses a significant threat for global crop production due to the absence of effective crop protection products. The aim of this study was to determine the virulence of R. solani and R. cerealis isolates towards Beta vulgaris, Zea mays, Triticum spelta and T. aestivum seedlings, to confirm the presence of endopolygalacturonase genes pg1 and pg5 in the genomes of the tested isolates and to evaluate the tested isolates’ sensitivity to triazole, strobilurin, imidazole and carboxamide fungicides. All tested isolates infected B. vulgaris seedlings. but none of them were virulent against Z. mays plants. R. solani isolates AG4 PL and AG2-2IIIB PL were characterized by the highest virulence (average infestation score of 2.37 and 2.53 points on a scale of 0–3 points) against sugar beet seedlings. The prevalence of infections caused by most of the analysed isolates (in particular R. solani AG4 J—11.8, and R. cerealis RC2—0.78) was higher in spelt than in bread wheat. The virulence of the analysed isolates was not correlated with the presence of pg1 and pg5 genes. The efficacy of the tested fungicides in controlling Rhizoctonia spp. infections was estimated at 100% (propiconazole + cyproconazole), 98.8% (penthiopyrad), 95.4% (tebuconazole) and 78.3% (azoxystrobin).

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.

Nivalenol‐producing Fusarium cerealis associated with fusarium head blight in winter wheat in Manitoba, Canada

Fusarium head blight (FHB) in small grain cereals is primarily caused by the members of the Fusarium graminearum species complex. These produce mycotoxins in infected grains, primarily deoxynivalenol (DON); acetylated derivatives of DON, 3‐acetyl‐DON (3‐ADON) and 15‐acetyl‐DON (15‐ADON); and nivalenol (NIV). This study reports the isolation of Fusarium cerealis in infected winter wheat heads for the first time in Canada. A phylogenetic analysis based on the TRI101 gene and F. graminearum species‐specific primers revealed two species of Fusarium: F. graminearum sensu stricto (127 isolates) and F. cerealis (five isolates). Chemotype determination based on the TRI3 gene revealed that 65% of the isolates were 3‐ADON, 31% were 15‐ADON and 4% were NIV producers. All the F. cerealis isolates were of NIV chemotype. Fusarium cerealis isolates can often be misidentified as F. graminearum as the morphological characteristics are similar. Although the cultural and macroconidial characteristics of F. graminearum and F. cerealis isolates were similar, the aggressiveness of these isolates on susceptible wheat cultivar Roblin and moderately resistant cultivar Carberry differed significantly. The F. graminearum 3‐ADON isolates were most aggressive, followed by F. graminearum 15‐ADON and F. cerealis NIV isolates. The findings from this study confirm the continuous shift of chemotypes from 15‐ADON to 3‐ADON in North America. In Canada, the presence of NIV is limited to barley samples and the discovery of NIV‐producing F. cerealis species in Canadian wheat fields may pose a serious concern to the Canadian wheat industry in the future.

Odporność na fungicydy izolatów Rhizoctonia cerealis

Sharp eyespot caused by Rhizoctonia cerealis is a common disease of cereals in Poland. R. cerealis is considered a weak pathogen, but under favourable conditions the incidence of the disease increases. The pathogen control is performed with fungicides used against other stem base diseases. The aim of the study was to evaluate the resistance level of R. cerealis isolates to selected fungicides that are used in the cereal crop protection. Cyproconazole and pyraclostrobin were fungicides that highly limited growth of the examined isolates. The group of the fungicides well-inhibiting growth of R. cerealis includes: epoxiconazole, flusilazole, tebuconazole, tetraconazole, azoxystrobin, carbendazim and next kresoxim-methyl and phenpropimorph. Most of the tested isolates were resistant to thiophanate-methyl, cyprodinil and prochloraz. The results indicate the possibility of effective control of R. cerealis by fungicides used for protection of other stem base diseases. Grzyb Rhizoctonia cerealis wywołujący ostrą plamistość oczkową jest częstym patogenem zbóż w Polsce. R. cerealis uważany jest za słabego patogena, lecz w sprzyjających warunkach występowanie choroby wzrasta. Grzyb zwalczany jest przy użyciu fungicydów stosowanych do ochrony upraw zbóż przed innymi patogenami podstawy źdźbła. Celem badań była ocena odporności izolatów R. cerealis na wybrane fungicydy stosowane w ochronie zbóż. Do związków bardzo dobrze ograniczających wzrost badanych izolatów zaklasyfikowane zostały cyprokonazol i piraklostrobina, do grupy dobrze hamującej wzrost R. cerealis: epoksykonazol, flusilazol, tebukonazol, tetrakonazol, azoksystrobina, karbendazym, następnie krezoksym metylu i fenpropimorf. Większość badanych izolatów R. cerealis była odporna na tiofanat metylu, cyprodinil i prochloraz. Wyniki wskazują na możliwość skutecznego ograniczania R. cerealis fungicydami przeznaczonymi do zwalczania innych chorób podstawy źdźbła.

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.

Genetic and Virulence Analysis of Rhizoctonia spp. Associated with Sugar Beet Root and Crown Rot in the Northeast Region of Iran

Rhizoctonia spp. are the main causal agents of root and crown rot on sugar beet. In this study, isolates of Rhizoctonia spp. were obtained from diseased sugar beet in Iran over 2 years. Of 68 isolates, 61 were R. solani and 7 were R. cerealis. The anastomosis group (AG) of all isolates was determined on glass slides against the testers. Characterization of intraspecific groups (ISGs) of R. solani isolates revealed that, of 61 isolates, 43 were AG2-2 IIIB and 18 were AG2-2 IV. Amplified fragment length polymorphism (AFLP) analyses were used to investigate genetic structure of Rhizoctonia populations. Principal coordinate plots and cluster analysis differentiated R. solani from R. cerealis isolates and separated the R. solani isolates belonging to different ISGs. AFLP data indicated that the R. solani and R. cerealis populations are not clonal. Analysis of molecular variance in AG2-2 IIIB isolates showed that geographic region was the main factor determining genetic structure of the populations. Sampling year had no significant effect on the genotypes. Pathogenicity tests on Beta vulgaris 'FD0432' revealed that R. solani AG2-2 IIIB and AG2-2 IV isolates were more virulent than R. cerealis.

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.

Formation of Oligosaccharides and Polysaccharides by Lactobacillus reuteri LTH5448 and Weissella cibaria 10M in Sorghum Sourdoughs

ABSTRACTGluten‐free breads, which are composed of gluten‐free flours, starch, and hydrocolloids, differ from wheat and rye breads in relation to texture, volume, and crumb structure. Moreover, the dietary fiber content is lower compared with wheat or rye breads. Cereal isolates of lactic acid bacteria frequently produce oligo‐ and homopolysaccharides from sucrose, which can improve the nutritional and technological properties of gluten‐free breads as prebiotic carbohydrates and hydrocolloids, respectively. Sorghum sourdough was fermented with Lactobacillus reuteri LTH5448 or Weissella cibaria 10M, which synthesize fructooligosaccharides (FOS) and levan, and isomaltooligosaccharides and dextran, respectively. The gluten‐free bread was produced with 14% sourdough addition. L. reuteri LTH5448 formed FOS and 1.5 g of levan/kg DM in quinoa sourdoughs. FOS were digested by the baker's yeast during proofing, and the levan could be qualitatively detected in the bread. W. cibaria 10M produced >60 g of isomaltooligosaccharides/kg DM and 0.6 g of dextran/kg DM, which could still be detected in the bread. Breads prepared with W. cibaria 10M were less firm compared with breads prepared with L. reuteri LTH5448 or a FOS and levan‐negative mutant of L. reuteri LTH5448. The addition of sourdoughs fermented with oligo‐ and polysaccharide forming starter cultures can increase the content of prebiotic oligosaccharides in gluten‐free breads.

Paecilomyces farinosus destroys powdery mildew colonies in detached leaf cultures but not on whole plants

Since 2001, several isolates of Blumeria graminis, the causal agent of cereal powdery mildew, maintained on detached leaves at the John Innes Centre, Norwich, UK, have spontaneously become infected with an unknown filamentous fungus whose mycelia have quickly overgrown the powdery mildew colonies and destroyed them completely. A total of five isolates of the contaminant were obtained and identified as Paecilomyces farinosus based on morphological characteristics and rDNA ITS sequence data. To determine whether these P. farinosus isolates can be considered as biocontrol agents (BCAs) of powdery mildews, we studied the interactions between P. farinosus and the following four powdery mildew species: B. graminis f.sp. hordei infecting barley, Oidium neolycopersici infecting tomato, Golovinomyces orontii infecting tobacco and Podosphaera fusca infecting cucumber. The powdery mildew colonies of all these four powdery mildew species were quickly destroyed by P. farinosus in leaf cultures but neither conidial suspensions nor cell-free culture filtrates of P. farinosus isolates could suppress the spread of powdery mildew infections on diseased barley, tomato, tobacco or cucumber plants in the greenhouse. It is concluded that P. farinosus cannot be considered as a promising BCA of powdery mildew infections although it can destroy powdery mildew colonies in detached leaf cultures and can be a menace during the maintenance of such cultures of cereal, apple, cucurbit and tomato powdery mildew isolates.

Variability and characterization of mycotoxin-producing Fusarium spp isolates by PCR-RFLP analysis of the IGS-rDNA region

In the present report, a total of 75 Fusarium spp isolates (35 of the Gibberella fujikuroi species complex, 26 of F. oxysporum, 7 of F. graminearum, 5 of F. culmorum, 1 of F. cerealis, and 1 of F. poae) from different hosts were characterized morphologically, physiologically and genetically. Morphological characterization was performed according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce fumonisin B1 (FB1), fumonisin B2 (FB2), zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). FB1, FB2, and ZEA were determined by liquid chromatography and trichothecenes by gas chromatography. Molecular characterization of isolates was carried out using an optimized and simple method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rDNA. The results indicated that G. fujikuroi complex isolates can be divided into low and high fumonisin producers. The haplotypes obtained with HhaI, EcoRI, AluI, PstI and XhoI enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin producing capacity. F. graminearum, F. culmorum and F. cerealis isolates were high ZEA and type B trichothecene producers, while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. The haplotypes obtained with CfoI, AluI, HapII, XhoI, EcoRI and PstI enzymes permitted to discern these five Fusarium species and G. fujikuroi complex isolates but the restriction patterns of the IGS region did not show any relationship with the geographic origin of isolates.

Characterization of Fusarium spp. isolates by PCR-RFLP analysis of the intergenic spacer region of the rRNA gene (rDNA)

In the present study, 44 Fusarium spp. isolates (5 Fusarium culmorum, 7 Fusarium graminearum, 1 Fusarium cerealis, 1 Fusarium poae, 26 Fusarium oxysporum, and 4 Gibberella fujikuroi species complex) were characterized morphologically, physiologically and genetically. All except one (Dutch Collection: CBS 620.72) were isolated from different hosts grown in various Spanish localizations. Morphological characterization was made according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). ZEA was determined by liquid chromatography and trichothecenes by gas chromatography. Confirmation was carried out by liquid chromatography-ion trap-mass spectrometry (ZEA) or gas chromatography-mass spectrometry (trichothecenes). Molecular characterization of isolates was performed using an optimized, simple and low-cost method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rRNA gene (rDNA). The results indicate that F. graminearum, F. culmorum and F. cerealis isolates were high ZEA and type B trichothecene producers, the F. poae isolate produced very low level of nivalenol while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. Restriction patterns of the IGS region did not show any relationship with the host, geographic origin of the isolate and mycotoxin-producing capacity. However, the haplotypes obtained with six restriction enzymes ( CfoI, AluI, HapII, XhoI, EcoRI and PstI) permitted to discern the six assayed Fusarium species. Therefore, this is a rapid and suitable methodology that allows closely related strains to group and to estimate the genetic relationships between the groups.

Genetic and pathogenic variation among cereal, medic and sub-clover isolates of Pythium irregulare

Genetic variation within 34 Pythium irregulare isolates was analyzed using restriction fragment length polymorphisms (RFLPs) as genetic markers. Most isolates had two alleles at several codominant RFLP loci and were scored as heterozygous. Heterozygotes were detected in F1 progeny from an in vitro cross and segregation ratios of the F2 progeny were not significantly different from those expected for allelic variation in a diploid. This confirmed that outcrossing occurs and contributes to genetic variation within the species. Phenetic analysis showed that isolates formed genetically related groups due to their host species and not due to similarities in their geographical origins. All isolates originating from medic formed a discrete group and were highly differentiated from the cereal and sub-clover isolates. The allelic distributions between isolates from these host-groups were significantly different. Most isolates also showed significant differences in their pathogenicity between hosts, indicating that they varied in pathogenic fitness and were better adapted to parasitising some hosts relative to others. These isolates were however, not necessarily more pathogenic on their host of origin. This research provided estimates of the extent of genetic and pathogenic diversity within P. irregulare and qualitative evidence for the occurrence of host-mediated selection and sexual outcrossing in the field.