THE REACTION OF WHEAT VARIETIES TO LOOSE SMUT AS DETERMINED BY EMBRYO, SEEDLING, AND ADULT PLANT TESTS

The possibility of using an embryo test as a means of determining the reaction of wheat varieties to loose smut ( Ustilago tritici (Pers.) Rostr.) has been investigated. Fifty-seven varieties were inoculated with two physiologic races by the partial vacuum method and the embryos, seedlings, and adult plants were examined for infection. Most of the varieties were fully susceptible to one or both races, and only ten showed real resistance to any one race. Braun R and Molinel proved almost immune to infection. The resistance in other varieties which showed no infection in the field was expressed as embryo susceptibility, i.e. the embryo became infected in much the same way as in field-susceptible varieties.
In the latter varieties infection passes from the embryo into the growing point of the young seedling. In the embryo-susceptible field-resistant varieties, infection does not pass from the scutellum and the growing point is therefore uninfected.
The reaction of most of the varieties tested was straightforward resistance or susceptibility, but in a few varieties a small proportion of the grains reacted differently from the majority. These reactions are discussed.     

THE DETERMINATION OF THE REACTION OF BARLEY VARIETIES TO LOOSE SMUT

The inoculation of barley with loose smut ( Ustilago nuda ) using various techniques has given results which do not reflect the known behaviour of varieties under natural conditions. No consistent results have been obtained, but all the varieties thus inoculated could become infected. No variety therefore possessed true physiological resistance and in the field some must escape infection. The inoculation techniques obviously overcame this escape.
A method has been devised which, with the varieties used, has reflected their behaviour in the field. Drops of coloured water or spore suspension are allowed to fall from a height on to ears at anthesis. Grains on the ears of varieties which do not become infected in the field do not become coloured or infected (depending on the drops used), while grains of varieties susceptible in the field do become coloured or infected. This is due to the glumes of the former group of varieties remaining closed, and so preventing access of smut spores under natural conditions.     

Some effects of barley yellow dwarf virus on spring and winter cereals in glasshouse trials

The tolerance of spring and winter varieties of wheat, oats and barley to infection by barley yellow dwarf virus (BYDV) was examined in glasshouse tests. Severely affected plants were stunted and grain yields were considerably decreased because of decreases in both ear number and numbers and sizes of grains. Winter barley varieties were very susceptible and many were killed by BYDV infection. The winter wheat varieties were more widely tolerant than those of oats and barley. Individual seedling symptoms, although correlated with reductions in yield, could not be relied upon for accurate classification of all varieties in order of their susceptibility to infection. Symptoms of seedling infection incorporated into an index of infection permit estimates to be made o eventual decreases in yield by applying the formula DY = 1.4 × (SH+LA+LL)+18. Thus decrease in grain yield (DY) can be related to decreases in height (SH) and leaf length (LL) and increases in leaf area discoloured (LA) in seedling plants infected with BYDV.     

甘蔗品种黑穗病抗性评价体系的建立

为了建立甘蔗品种黑穗病抗性评价体系,选用9个引进品种,设计一个包括6个对照品种在内的田间试验。首次采用混合小种进行人工浸渍接种,通过整个新植蔗生长季病害进展曲线下的面积、茎感染率和株感染率3个病情指数,以及病害流行学参数潜伏侵染期和持续发病期的分析,对其扰痛性进行评价。在分析以上参数相关程度的基础上,引进了系统聚类分析法进行进一步评价。结果显示;9个引进甘蔗品种中,ROC26属于感病品种,其余属于抗病或高抗品种;3个病情指数和持续发病期的两两相关均为显著正相关,潜伏侵染期与这些参数的相关为负相关,但未达到显著水平;在类间距离大于1．0的条件下,可将15个品种聚为6类,进一步明确了备品种抗黑穗痛性的相似程度;抗性鉴定标准对照种NC0310、F134、NC0376和Ya71—374的应用,明确了接种源为小种1和小种2,通过一次接种试验,明确了供试品种对2个小种的抗性水平,标准对照种的抗性表现,还说明了本生长季发病条件基本是适宜的。本文建立的甘蔗品种抗黑穗病评价体系是切实可行的。     

LOOSE SMUT (USTILAGO TRITICI (PERS.) ROSTR.) OF WHEAT: PHYSIOLOGIC SPECIALIZATION AND REACTION OF VARIETIES IN ENGLAND

Three physiologic races of loose smut (named Ci, C2 and C3), found in wheat varieties grown at Cambridge, differ from those identified in Holland. The resistance or susceptibility of fifty-seven wheat varieties to races Ci, C2 and C3 has been determined. Experiments have shown that infection of an ear can take place over a period of several days. There is a close relationship between the percentage of plants attacked and the severity of attack in a single plant.     

The application of real-time PCR to the identification, detection and quantification of Pyrenophora species in barley seed

A real-time quantitative PCR technique has been used to develop a rapid and sensitive seed health test for Pyrenophora spp. on barley seed. Using the fluorescent reporter dye SYBR Green I for real-time detection of PCR amplification, pathogen DNA extracted from infected seed can be quantified to the picogram level. The amount of Pyrenophora DNA extracted from seed samples of an artificial infection level gradient, constructed by mixing infected and uninfected seed, correlated with good agreement ( r  = 0.931) to percentage infection levels of the same samples measured by agar plate testing. In addition, a correlation of r  = 0.883 was obtained between the two testing methods for naturally infected seed, ranging from 0% to 89% infection. Samples could be quantified to below the 2% voluntary threshold required for deciding on seed treatment. The proposed test was performed in three parts: (i) quantification of Pyrenophora spp. infection using Pyrenophora -specific PCR primers; (ii) test of any negative samples from (i) with barley-specific PCR primers to check the DNA extraction process; (iii) test of positive samples from (i) for the presence of Pyrenophora graminea using P. graminea -specific PCR primers. All PCRs were performed in the LightCycler™ instrument allowing each PCR run and analysis to be completed within 30 min. With the current daily receipt of samples (batches up to 16) the test can be completed in 8 h, compared to 7 days for the traditional agar plate test.     

THE INCIDENCE OF CERTAIN SEED-BORNE DISEASES IN COMMERCIAL SEED SAMPLES

The incidence of ergot, Claviceps purpurea (Fr.) Tul., in commercial cereal seed samples submitted to the Official Seed Testing Station from 1918 to 1957 has been examined.
Rye samples were more frequently contaminated than wheat or barley and no cereal ergots were recorded in oats.
A relatively high proportion of ergot-contaminated rye samples occurred every 8–10 years. This appeared to be associated with high relative humidity and low maximum temperature during June. Similar trends were found for wheat and barley.
No varietal differences in susceptibility to ergot contamination were found for rye or barley, but there was some evidence that spring wheat varieties were more frequently contaminated than winter ones.     

Presence of caprine arthritis-encephalitis virus (CAEV) proviral DNA in genital tract tissues of superovulated dairy goat does

Transmission of caprine arthritis-encephalitis virus (CAEV) is not completely understood and the vertical route of infection from the goat to the embryo or to the fetus needs to be investigated. This route of infection involves the presence of CAEV in the genital tract tissues. Prior studies have detected CAEV-infected cells in genital secretions and in flushing media recovered during embryo collection from infected goats. To specify the origin of these cells, we conducted a double-nested polymerase chain reaction (PCR) test on embryo flushing media and on mammary gland, mammary lymph node, synovial membrane, pelvic lymph node, uterus and oviduct tissues from 25 CAEV-infected (blood PCR positive) embryo donor goats for the presence of CAEV proviral DNA.The presence of proviral DNA was found in 22 of 25 mammary gland samples, 14 of 25 uterus samples, and in 16 of 25 oviduct samples. Nineteen of 25 goats had at least one positive genital tract sample. Flushing media from 11 goats were PCR positive. All goats with positive-flushing media were oviduct positive. Of this group of does, except for 1 of the 11, infection of flushing media correlated with infection of almost all the other tissues examined. The frequency of positive tissues for flushing media-positive goats (61/66; 92%) was significantly higher than that for flushing media-negative goats (50/84; 60%) (P<0.01).This study demonstrated the presence of CAEV-infected cells in the goat genital tract. The presence of CAEV-infected cells in the uterus and oviducts suggests potential for vertical transmission of CAEV from doe to embryo or fetus.     

Influence of brown stink bug feeding,planting date and sampling time on common smut infection of maize

Phytopathogen infections are frequently influenced by both biotic and abiotic factors in a crop field. The effect of brown stink bug, Euschistus servus (Hemiptera: Pentatomidae), feeding and planting date and sampling time on common smut (Ustilago maydis) infection percentage of maize plants was examined in 2005 and 2006, and 2010 and 2011, respectively. Brown stink bug adult feeding on maize hybrid “DKC6971” at flowering in 2005 and 2006 did not influence smut infection percentage when examined using 3 treatments (i.e., 0 adult, 5 adults, and 5 adults mixed with the smut spores). The smut infection percentages were <3% (n = 12) in the 3 treatments. The smut infection percentage among the 4 weekly samplings was the same, so was natural aflatoxin contamination at harvest among the treatments. The 2nd experiment showed that planting date did not affect the smut infection percentage in either 2010 or 2011. But, the smut infection percentage from the postflowering sampling was greater than preflowering sampling in both years. The smut infection percentage varied among the germplasm lines in 2010, but not in 2011. This study demonstrated that brown stink bug feeding at flowering had no effect on smut infection in maize, and the best time for smut evaluation would be after flowering. The temperature and precipitation might have also influenced the percentage of smut‐infected maize plants during the 4 years when the experiments were conducted. The similarity between kernel‐colonizing U. maydis and Aspergillus flavus infections and genotype × environment interaction were also discussed.     

Targeted development of a microsatellite marker associated with a true loose smut resistance gene in barley (Hordeum vulgare L.)

Microsatellite markers have many of the properties of an ideal marker, but development of microsatellite markers is tedious, time-consuming and expensive. In the past few years, great efforts have been made to develop, map and utilize microsatellite markers in various crops. It is still a major challenge to find a microsatellite marker associated with an economically important trait. In the present study we report on the targeted development of a microsatellite marker to a barley disease resistance gene. The method includes the following steps: (1) pooling DNA samples from a segregating population based on the principle of bulked-segregant analysis; (2) digesting the pooled DNAs and ligating adaptors; (3) selectively amplifying and identifying polymorphic microsatellites; and (4) developing primers for the microsatellite associated with the targeted trait. Using this method, a microsatellite marker associated with the true loose smut resistance gene (Un8) in the Harrington × TR306 doubled-haploid population was identified. This marker showed polymorphism in four breeding populations segregating for true loose smut resistance. In three of these populations, genetic distance between the microsatellite and the true loose smut resistance gene varied from 8.6 to 10.3 cM. Polymorphism of the microsatellite was tested among three disease resistant lines and 21 susceptible cultivars. Fourteen to eighteen of the 21 susceptible cultivars exhibited a polymorphism for the microsatellite with respect to at least one of the disease-resistant lines. This method for the targeted development of microsatellite markers should have widespread applicability and should efficiently provide highly polymorphic markers for use in breeding programs.     

Hypoxia interferes with ABA metabolism and increases ABA sensitivity in embryos of dormant barley grains

Two mechanisms have been suggested as being responsible for dormancy in barley grain: (i) ABA in the embryo, and (ii) limitation of oxygen supply to the embryo by oxygen fixation as a result of the oxidation of phenolic compounds in the glumellae. The aim of the present work was to investigate whether hypoxia imposed by the glumellae interferes with ABA metabolism in the embryo, thus resulting in dormancy. In dormant and non-dormant grains incubated at 20 degrees C and in non-dormant grains incubated at 30 degrees C (i.e. when dormancy is not expressed), ABA content in the embryo decreased dramatically during the first 5 h of incubation before germination was detected. By contrast, germination of dormant grains was less than 2% within 48 h at 30 degrees C and embryo ABA content increased during the first hours of incubation and then remained 2-4 times higher than in embryos from grains in which dormancy was not expressed. Removal of the glumellae allowed germination of dormant grains at 30 degrees C and the embryos did not display the initial increase in ABA content. Incubation of de-hulled grains under 5% oxygen to mimic the effect of glumellae, restored the initial increase ABA in content and completely inhibited germination. Incubation of embryos isolated from dormant grains, in the presence of a wide range of ABA concentrations and under various oxygen tensions, revealed that hypoxia increased embryo sensitivity to ABA by 2-fold. This effect was more pronounced at 30 degrees C than at 20 degrees C. Furthermore, when embryos from dormant grains were incubated at 30 degrees C in the presence of 10 microM ABA, their endogenous ABA content remained constant after 48 h of incubation under air, while it increased dramatically in embryos incubated under hypoxia, indicating that the apparent increase in embryo ABA responsiveness induced by hypoxia was, in part, mediated by an inability of the embryo to inactivate ABA. Taken together these results suggest that hypoxia, either imposed artificially or by the glumellae, increases embryo sensitivity to ABA and interferes with ABA metabolism.     

Interactions of Polyoma and Mouse DNAs. IV. Time Course and Extent of Integration of Polyoma DNA into Mouse DNA During Lytic Infection

The time course of covalent binding of polyoma viral DNA to mouse DNA was followed in mouse embryo cells that had been grown prior to infection in the presence of 5-bromodeoxyuridine. Density-labeled (HL) mouse DNA was separated from free polyoma DNA by CsCl isopycnic centrifugation. Polyoma DNA sequences present in HL mouse DNA were detected by hybridization with radioactive cRNA synthesized in vitro. In reconstruction experiments, the limit of detection was found to be, on the average, about 0.5 genome equivalent (g.e.) of polyoma DNA per cell. To find conditions for the isolation of HL mouse DNA and for its complete separation from free polyoma DNA, cultures infected at 4 degrees C were used. HL mouse DNA extracted with sodium dodecyl sulfate and high salt concentrations (5 to 6 M CsCl) and then purified by three consecutive CsCl density gradient centrifugations was free from detectable amounts of polyoma DNA, whereas HL mouse DNA extracted with chloroform and phenol and purified in the same way always contained contaminating, noncovalently bound polyoma DNA. In lytically infected bromodeoxyuridine-prelabeled mouse embryo cultures, polyoma DNA bound to HL mouse DNA that had been extracted by the sodium dodecyl sulfate-CsCl procedure was first detected in small amounts (1 to 2 g.e. per cell) at 10 h after infection. In cultures incubated with medium containing thymidine (5 mug/ml), 4 to 6 g.e. of polyoma DNA per cell was detected at 14 and 18 h after infection. In these samples, practically all viral DNA was bound to high-molecular-weight HL mouse DNA. In cultures incubated with normal medium (no additions) and extracted between 17 and 20 h after infection, 20 to 350 g.e. of polyoma DNA per cell banded with HL mouse DNA. However, when DNA of one of these samples was subfractionated by sodium dodecyl sulfate-salt precipitation prior to isolation of HL mouse DNA, about 80% of the viral DNA banding at increased density was present in the low-molecular-weight DNA fraction. This observation suggests that in normal medium some progeny viral DNA of increased density was synthesized. Covalent binding of polyoma DNA to density-labeled mouse DNA was demonstrated by alkaline CsCl density gradient centrifugation: nearly equal amounts of polyoma DNA were found in the H and L strands, respectively, as is expected for linear integration of viral DNA. The results lead to the conclusions that (i) early polyoma mRNA is transcribed from free parental viral DNA; (ii) covalent linear integration is first detectable at the time when tumor (T)-antigen is synthesized; and (iii) only few copies (<10 g.e./cell) become integrated between 10 and 18 h after infection, i.e., during the period when cellular and viral DNA replication starts in individual cells.     

Mechanism of Oncogenic Transformation by Rous Sarcoma Virus: I. Intracellular Inactivation of Cell-Transforming Ability of Rous Sarcoma Virus by 5-Bromodeoxyuridine and Light

Chick embryo fibroblasts brought into stationary phase of growth by maintenance in serum-free Eagle's MEM medium were infected with the Bryan strain of Rous sarcoma virus (B-RSV) and incubated for 18 hr in the presence of 5-bromo-deoxyuridine (BUdR). The cells were then allowed to resume growth and deoxyribonucleic acid (DNA) synthesis by addition of an enriched F12 medium containing serum and RSV antibody to prevent spread of viral infection. After 48 hr, the cultures were exposed for various periods to visible light, overlaid with solid culture medium, and observed for the appearance of foci of transformed cells. In cultures treated with BUdR at the time of infection, exposure to light resulted in a suppression of focus formation of from 50 to 90% in various experiments. Treatment with BUdR for 18 hr before infection or on the day after infection, followed by exposure to light, had no effect on focus formation. In cultures in which almost all cells were infected, treatment with BUdR followed by exposure to light did not result in cell death. This suggests that suppression of transformation is not due to selective killing of infected cells by this treatment but rather to the intracellular inactivation of the transforming ability of Rous sarcoma proviral DNA.     

稻曲球及稻曲病菌菌落微结构的SEM观察

本文对不同培养条件下稻曲病菌菌落及稻曲球的微结构进行了扫描电镜比较研究。在PS培养液里进行液体培养时,稻曲病菌很少产生分生孢子和厚垣孢子,只有培养后期漂浮在培养液表面的菌落可以产生大量的厚垣孢子。病原菌在进行PSA固体培养时,大部分菌株在培养后期产生大量的成堆分布的厚垣孢子,少部分菌株在菌落上产生散生的厚垣孢子。说明暴露于空气有助于稻曲病菌产生厚垣孢子。在煮熟的带壳谷粒上稻曲病菌的生长明显比在去壳上的要慢得多。微结构分析表明,稻曲球表面是一层密集的厚垣孢子,菌丝与稻粒的胚乳层界限分明,大部分稻曲球中部有大块的发育良好的胚乳,并充满密集的淀粉粒。说明稻曲病菌可能在开花灌浆后开始侵染,而且至少后期是腐生的。     

稻曲球及稻曲病菌菌落微结构的SEM观察

本文对不同培养条件下稻曲病菌菌落及稻曲球的微结构进行了扫描电镜比较研究。在PS培养液里进行液体培养时,稻曲病菌很少产生分生孢子和厚垣孢子,只有培养后期漂浮在培养液表面的菌落可以产生大量的厚垣孢子。病原菌在进行PSA固体培养时,大部分菌株在培养后期产生大量的成堆分布的厚垣孢子,少部分菌株在菌落上产生散生的厚垣孢子。说明暴露于空气有助于稻曲病菌产生厚垣孢子。在煮熟的带壳谷粒上稻曲病菌的生长明显比在去壳上的要慢得多。微结构分析表明,稻曲球表面是一层密集的厚垣孢子,菌丝与稻粒的胚乳层界限分明,大部分稻曲球中部有大块的发育良好的胚乳,并充满密集的淀粉粒。说明稻曲病菌可能在开花灌浆后开始侵染,而且至少后期是腐生的。     

Studies on the Germination of Cereals: 2. Factors Determining the Germination Behaviour of Wheat Grains during Maturation

A comparative study has been made of the germination behaviourof a white-grained and a red-grained wheat variety throughoutthe course of maturation. Although freshly harvested wheat grains do not germinate duringthe early stages of ripening, the embryo will develop undergermination conditions when it is completely exposed, or theepidermis of the pericarp only is removed. At the harvest-ripestage the white grains germinate rapidly, but the emergenceof the embryo is considerably delayed in the red grains. Thevarietal difference is due to the presence of the covering layersand becomes increasingly apparent in grains which have beenprematurely harvested, between 3 and 6 weeks after anthesis,and then subjected to desiccation. This effect of the coveringlayers on embryo development does not appear to be related totheir permeability to water or oxygen. The behaviour of thetwo wheat varieties is thus determined by the mechanical propertiesof the covering layers, and especially the pericarp. These delaygermination in the red grains until there has been an increasein the water absorbing power of the imbibed embryo.     

Influence of different carbohydrate composition in barley varieties on Salmonella Typhimurium var. Copenhagen colonisation in a "Trojan" challenge model in pigs

Based on previously performed in vitro studies, which showed that hulless barley varieties could reduce large intestinal Salmonella Typhimurium var. Copenhagen proliferation in pigs, two in vivo experiments were conducted to prove these observations. In Experiment (Exp.) 1, 126 weaning piglets were randomly allocated into pens of seven animals each and fed one of six experimental diets. Three diets contained (75% as-fed) one of three hulless barley varieties with beta-glucan (BG) contents ranging from 5 to 11% and amylose from 5 to 40%, and two diets contained a low BG and amylose hulless barley supplemented with isolated barley BG or raw potato starch. A hulled barley diet served as a control. Two piglets per pen ("Trojan" pigs) were orally infected with Salmonella Typhimurium var. Copenhagen (ST). The remaining five pigs per pen were designated "Contact" pigs. The ST shedding was determined over one week after infection. On day 6, the two Trojans and two random Contacts from each pen were euthanised and intestinal contents and mesenteric lymph nodes cultured for ST. Intestinal volatile fatty acids and microbial composition were determined. In Exp. 2, 126 piglets were assigned to one of three diets based on hulled or hulless barleys. The timeline, infection, sampling and analyses were similar as in Exp. 1 except samples were taken from four Contact pigs. Hulless barley varieties with high BG and amylose tended to decrease ST persistence in Exp. 1. Clostridia from cluster I in the colon were reduced with high amylose hulless barley or diets supplemented with potato starch (p < 0.05), whereas other microbial groups were not. Propionate increased (p < 0.05) and acetate decreased (p < 0.05) with hulless barley inclusion. Exp. 2 revealed a reduced ST shedding and reduced number of clostridia for high BG hulless barley as compared to common hulled barley and a low BG variety (p < 0.05). In conclusion, high BG hulless barley do not prevent ST colonisation but might help to reduce transmission in pigs, likely by supporting an intestinal environment limiting growth of this zoopathogen.     

BACTERIAL CANKER OF STONE-FRUITS: IV. INVESTIGATION OF A METHOD FOR MEASURING THE INOCULUM POTENTIAL OF CHERRY TREES

Pseudomonas mors-prunorum Wormald, the organism causing bacterial canker of stone-fruits, was present on healthy cherry leaves during the autumn in numbers sufficient to suggest that they were the main source of inoculum for the infection of stems and branches. Quantitative estimates of the pathogen could be obtained by shaking leaves in water and plating out the washings. This method was examined in detail as a possible means of comparing the inoculum potentials on different cherry varieties.
The numbers of the pathogen varied considerably between leaves and between branches, but not between trees of the same variety. It was estimated that a sample of 192 leaves, eight from each of twenty-four different branches would be adequate to show differences between varieties if sufficiently replicated in time. The accuracy of a twenty-four branch sample was tested by comparing parallel samples from two separate groups of trees of the same variety. There was a highly significant correlation between the numbers of bacteria obtained from the two series of samples. From the error variance in an analysis of variance, it was calculated that five successive samples would show a significant difference between two such groups of trees if one exceeded the other by 54%.
It was necessary to wash leaves for 4 hr. or more, depending on temperature, to recover all the bacteria from the leaf surfaces. At temperatures lower than 27° C. however, bacterial growth intervened before this stage was reached. At laboratory temperatures a period of 4 hr. washing was found to be most suitable for comparing varieties and gave 80–90% recovery of bacteria without errors due to growth.
A characteristic flora of non-parasitic bacteria was observed on cherry trees, coexisting with the pathogen on the leaf surfaces. The fungus Pullaria pullulans was also extremely abundant.     

The impact of Fusarium culmorum infection on the protein fractions of raw barley and malted grains

Contaminating fungi, such as Fusarium species, produce metabolites that may interfere with normal barley grain proteolysis pattern and consequently, affect malt and beer quality. Protein compositional changes of an initial mixture of 20 % Fusarium culmorum infected and 80 % noninfected mature barley grains and respective malt are reported here. Proteolytic activity of infected barley grains (IBG) and respective malt, with controls (uninfected grains), were characterized using protease inhibitors from each class of this enzyme, including metallo-, cysteine, serine, and aspartic proteases, as well as uninhibited protease fractions. The proteins were extracted according to the Osborne fractionation and separated by size exclusion chromatography. Additionally, two-dimensional (2D) gel electrophoresis (GE) was used to analyze hydrophobic storage proteins isolated from the control and IBG. Analyses revealed that F. culmorum IBG had a twofold increase of proteolytic activity compared to the control sample, which showed an increase in all protease classes with aspartic proteases dominating. Infected and control malt grains were comparable with cysteine proteases representing almost 50 % of all proteolytic enzymes detected. Protein extractability was 31 % higher in IBG compared to the control barley. The albumin fraction showed that several metabolic proteins decreased and increased at different rates during infection and malting, thus showing a complex F. culmorum infection interdependence. Prolamin storage proteins were more hydrophobic during barley fungal infection. F. culmorum interfered with the grain hydrolytic protein profile, thereby altering the grain's protein content and quality.