浙江柑橘黄龙病病原β-操纵子核糖体蛋白基因的PCR检测及其序列比对

长期的观察发现,不同柑橘品种感染黄龙病后出现的病症也不尽相同,针对这种现象本文检测了浙江柑橘黄龙病病原β-操纵子核糖体蛋白基因并进行了序列比对,BLAST比对结果说明所扩增的基因序列之间差异性为0,与基因库(NCBI)中黄龙病亚洲韧皮杆菌DNA序列相似性为100％,由此说明浙江柑橘黄龙病病原β-操纵子核糖体蛋白基因并未发生变异,出现上述现象可能是其他基因发生变异或者与品种的抗病性有关。     

广东三个柑橘品种不同生育期的黄龙病菌含量动态变化研究

采用实时荧光定量PCR检测了广东省3个柑橘品种在不同生育期的黄龙病菌含量,探索其动态变化规律,为病害防控提供依据。结果表明,第一年,3个品种春梢期的黄龙病菌平均含量分别为3.52×10~5、4.76×10~5、1.03×10~5拷贝/g,显著低于相应夏梢期(5.50×10~5、9.48×10~5、2.50×10~5拷贝/g)和秋梢期(5.32×10~5、9.84×10~5、2.59×10~5拷贝/g);夏梢期与秋梢期含量又显著低于相应冬梢期(21.60×10~5、17.65×10~5、6.86×10~5拷贝/g)及果实成熟期(14.57×10~5、16.45×10~5、7.96×10~5拷贝/g);砂糖橘全年黄龙病菌含量与贡柑无显著差异,但均显著高于柠檬;第二年的数据与第一年类似,但砂糖橘和贡柑在多个生育期的含菌量比上年均显著升高,而柠檬所有生育期菌量均无显著变化。结果显示:柑橘病树内的黄龙病菌含量在春梢期最低,夏梢、秋梢期次之,冬梢期及果实成熟期最高;‘柠檬’相对耐病,体现为含菌量相对较低、增速较慢。     

Note: Comparison of three laboratory methods to evaluate the pathogenicity and virulence of tenPseudomonas syringae pv.syringae strains on apple, pear, cherry and peach trees

The pathogenicity and virulence of ten GreekPseudomonas syringae pv.syringae strains from different hosts (citrus, pear, apple, peach and cherry) were evaluated using three different laboratory methods, which produced results in good agreement. All ten strains were virulent on apple, pear, cherry and peach trees. The extent of tissue colonized varied considerably among strains and cultivars. On excised shoots and twigs of apple and pear, strains BPI 176, BPI 203, PI 2 and PI 14 were the most virulent and strains BPI 689, BPI 992, BPI 4, BPI 20, PI 18 and PI 19 were the least virulent. On excised shoots and twigs of peach and cherry, strains BPI 176, BPI 203, PI 2, PI 14, PI 18 and PI 19 were the most virulent and strains BPI 4 and BPI 20 were the least virulent. Moderate virulence was evinced by strains BPI 689 and BPI 992. These pathogenicity assays are proposed as rapid and reproducible screening systems to evaluate the susceptibility of apple, pear, cherry and peach cultivars to this bacterial pathogen.     

6种柑橘类植物对柑橘衰退病毒分离株TR-L514变异的影响

 柑橘衰退病毒(Citrus tristeza virus,CTV)存在复杂的株系分化现象,运用弱毒株交叉保护防治柑橘衰退病时需了解不同类型柑橘对CTV构成的影响。作者将CTV分离株TR-L514嫁接接种到6类柑橘植物上获得了18个亚分离株,并对这18个亚分离株和TR-L514进行了指示植物鉴定,p25基因的限制性片段长度多态性(RFLP)和单链构象多态性(SSCP)分析,p23基因的序列比较。结果表明,CTV株系对不同柑橘类植株的适应性存在差异。TR-L514嫁接到不同柑橘类植株其构成会发生变化,在甜橙上可以同时检测出p25//HinfⅠ RFLP第1和6组群,并具有比在其它4类柑橘上更为复杂的SSCP谱型构成,因此甜橙可能更适宜于CTV的增殖。TR-L514及18个亚分离株的p23基因序列差异可能与不同类柑橘植株的适应性有关。     

Laboratory Evaluation of Citrus Cultivars Susceptibility and Influence of Fruit Size on Fortune Mandarin to Infection by Alternaria alternata pv. citri

Young leaves of 62 citrus cultivars were inoculated with conidia of three Spanish isolates of Alternaria alternata pv. citri, the causal agent of brown spot of citrus. Hybrids with Dancy mandarin, King mandarin or their derivates as a parent, grapefruit cultivars and the mandarin cultivars Guillermina, Emperor, Clemenpons and Esbal were highly susceptible to the pathogen. Satsuma cultivar Clausellina and orange cultivars, with the exception of Sanguinelli, were slightly susceptible. Lemon and lime cultivars were not susceptible, with the exception of Mexican lime (Citrus aurantifolia), which was slightly susceptible. Although this study shows a range of potential hosts for this pathogen, to date the only affected cultivars in Spain are Fortune and Nova mandarins, and Minneola tangelo. The susceptibility of Fortune fruits decreased as diameter increased, being susceptible through the whole season. This was confirmed with field observations in autumn where fruit infections have been detected when the diameter reaches 6–7 cm.     

Ultrastructural and immunocytochemical investigation of pathogen development and host responses in resistant and susceptible wheat spikes infected by Fusarium culmorum

Pathogen development and host responses in wheat spikes of resistant and susceptible cultivars infected by Fusarium culmorum causing Fusarium head blight (FHB), were investigated by means of electron microscopy as well as immunogold labelling techniques. The studies revealed similarities in the infection process and the initial spreading of the pathogen in wheat spikes between resistant and susceptible cultivars. However, the pathogen’s development was obviously more slow in the resistant cultivars as in comparison to a susceptible one. The structural defence reactions such as the formation of thick layered appositions and large papillae were essentially more pronounced in the infected host tissues of the resistant cultivars, than in the susceptible one. β -1,3-glucan was detected in the appositions and papillae. Furthermore, immunogold labelling of lignin demonstrated that there were no differences in the lignin contents of the wheat spikes between susceptible and resistant cultivars regarding the uninoculated healthy tissue, but densities of lignin in host cell walls of the infected wheat spikes differed distinctly between resistant and susceptible cultivars. The lignin content in the cell walls of the infected tissues of the susceptible wheat cultivar increased slightly, while the lignin accumulated intensely in the host cell walls of the infected wheat spikes of the resistant cultivars. These findings indicate that lignin accumulation in the infected wheat spikes may play an important role in resistance to the spreading of the pathogen in the host tissues. Immunogold labelling of the Fusarium toxin DON in the infected lemma showed the same labelling patterns in the host tissues of resistant and susceptible cultivars. However, there were distinct differences in the toxin concentration between the tissues of the susceptible and resistant cultivars. At the early stage of infection, the labelling densities for DON in resistant cultivars were significantly lower than those in the susceptible one. The present study indicates that the FHB resistant cultivars are able to develop active defence reactions during infection and spreading of the pathogen in the host tissues. The lower accumulation of the toxin DON in the tissues of the infected spikes of resistant cultivars which results from the host’s defence mechanisms may allow more intensive defence responses to the pathogen by the host.     

Molecular detection and quantification of Colletotrichum abscissum in sweet orange propagative material

Postbloom fruit drop disease (PFD) has caused serious impacts on citrus production in the Americas, occurring sporadically and suddenly during rain in the flowering period. In São Paulo State, Brazil, Colletotrichum abscissum is the causal agent responsible for over 80% of the disease incidence. Pathogen dispersal over long distances and the origin of primary inoculum are still unclear for PFD epidemics. We tested the hypothesis that citrus propagation material can harbour C. abscissum DNA by quantifying it in leaves of budwood increase block trees (BIB) and young citrus plants (YP) using multiplex quantitative PCR (qPCR). C. abscissum DNA was detected in all citrus nurseries, regardless of the type of propagative material, the sweet orange variety, or the nursery location. Overall, 73.4% of all samples from citrus nurseries have DNA from the pathogen, with a detection limit of 10 conidia. The average of 155 conidia found in YP was higher than the conidia observed on leaf samples from BIB (p = 0.03), although leaf samples from cultivars Valencia and Pera did not differ significantly, with means around 127 and 118 conidia, respectively (p = 0.75). This is the first molecular detection of C. abscissum in citrus propagative material. The multiplex qPCR assay may be used as a protocol for an accurate diagnosis of C. abscissum in citrus propagative material, may assist a better understanding of the pathogen dispersal over long distances, and may be used for further studies involving the quantification of C. abscissum in citrus orchards.     

LncRNA在柑橘木虱与黄龙病病原菌互作中的差异表达分析及验证

为明确柑橘黄龙病的唯一自然传播媒介——柑橘木虱Diaphorina citri的长链非编码RNA(long non-coding RNA,lncRNA)是否参与调控黄龙病病原菌Candidatus Liberibacter asiaticus(CLas)的侵染及复制,采用生物信息学预测及PCR扩增方法进行lncRNA的预测、特征分析、验证及差异表达分析。结果显示,柑橘木虱的13个转录组RNA-Seq数据中共有10 192个lncRNA基因,对应15 747条lncRNA转录本;与蛋白质编码基因相比,柑橘木虱lncRNA具有更少的外显子数量和更短的转录本长度;随机选取的10条lncRNA基因中,有7条lncRNA基因在无菌柑橘木虱广州品系或赣州品系中有表达,其中1条lncRNA基因TCONS_00034665在无菌广州品系和无菌赣州品系中存在差异表达;带菌和无菌柑橘木虱成虫中预测获得2个差异表达的lncRNA基因TCONS_00096118和TCONS_00234564,实时荧光定量PCR验证发现TCONS_00234564与预测结果一致,在带菌柑橘木虱成虫中高表达。表明lncRNA参与了黄龙病病原菌与寄主柑橘木虱的互作。     

Modes of Action of Pantoea agglomerans CPA-2, an Antagonist of Postharvest Pathogens on Fruits

Pantoea agglomerans CPA-2 is an effective antagonist against the postharvest pathogens Penicillium digitatum and Penicillium italicum on citrus fruits but its mode of action is unknown. Possible mechanisms studied in this work were antibiosis, induced resistance, competition and production of chitinolytic enzymes. P. agglomerans CPA-2 was unable to produce antibiotics or chitinolytic enzymes under the conditions tested. Induction of resistance by P. agglomerans CPA-2 was studied in oranges by measuring phenylalanine ammonia lyase and peroxidase enzyme activity in the orange peel at different time points after inoculation with the antagonist and/or the pathogen. No significant augmentation of enzyme activity after inoculation of oranges with P. agglomerans CPA-2 in the presence or absence of the pathogen was observed. P. agglomerans was effective only when it is in close contact with the pathogens. Competition for nutrients was studied using tissue culture plates with cylinder inserts, which allowed competition for nutrients to be studied without competition for space since physical contact between pathogen and antagonist was avoided. The presence of P. agglomerans in the tissue culture wells clearly decreased the germination of Penicillium conidia present in the cylinder when diluted orange peel extract or diluted potato dextrose broth was the nutrient source. Germination of Penicillium conidia, however, was almost completely inhibited when pathogen and antagonist were in physical contact. These results indicate that competition for nutrients is one of the modes of action of P. agglomerans CPA-2, but that physical contact between pathogen and antagonist is important for effective control.     

Development of real-time PCR systems based on SYBR? Green I and TaqMan? technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus

Real-time PCR assays based on SYBR? Green I and TaqMan? technologies were developed for in planta detection and quantification of Phoma tracheiphila, the mitosporic fungus causing ‘mal secco’ disease on citrus. Primers and a hybridization probe were designed on the basis of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The real-time PCR assays were compared with a classic isolation method in two separate experiments carried out on 6 and 24 month-old sour orange seedlings, artificially inoculated with a conidial suspension of the pathogen. Both technologies made it possible to follow the progression of infection by P. tracheiphila, enabling detection and quantification of the target fungus prior to the development of symptoms. The detection limit was 10 copies of the cloned target sequence and 15 pg of genomic DNA extracted from fungal spores. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the target DNA, indicating that the method is suitable as a qualitative and quantitative assay. The presence of non-target fungal DNA had no effect on the specificity of the assay, but resulted in a 10-fold reduction of sensitivity. Total inhibition of the reaction occurred when conidia of the target pathogen were mixed with an organic soil substrate before extracting DNA using the standard protocol, while an alternative purification kit resulted in a significant decrease in sensitivity. Compared to classic methods, real-time PCR proved faster and easier to perform and showed a higher sensitivity. These results suggest that real-time PCR, based on both chemistries, has a great potential for early diagnosis of ‘mal secco’ disease and for quantitative estimation of fungal growth within host tissue.     

Regulation of defense-related enzymes associated with bacterial spot resistance in Tomato

Twenty tomato (Solanum lycopersicon) cultivars were screened for resistance against bacterial spot disease incited byXanthomonas axonopodis pv.vesicatoria under field conditions with and without pathogen infection. Screening was done by artificially inoculating aX. axonopodis pv.vesicatoria suspension to 4-week-old tomato seedlings and observing them for typical symptoms of the disease. Seedlings were categorized into highly resistant, resistant, susceptible and highly susceptible cultivars on the basis of disease incidence. Tomato cultivars were screened for defense-related enzymes, total phenols and lignin contents. The temporal patterns of all these enzymes were estimated with a moderately susceptible tomato cultivar. Native PAGE analysis of both peroxidase (POX) and polyphenol oxidase (PPO) was carried out for the time course of enzyme activities and also by selecting three different tomato cultivars, following infection with the pathogen. Based on the inducible amounts of these enzymes upon pathogen infection, the tomato cultivars were correlated with the disease incidence under field conditions. A significant (P≤0.05) correlation was observed between the degree of host resistance and the enzyme levels. In highly resistant tomato cultivars the enzyme levels, total phenols and lignin contents were increased in comparison with highly susceptible tomato cultivars. Isoform analysis of POX and PPO enzymes indicated a clear difference between resistant and susceptible tomato cultivars in the number of isoforms and also in the intensity of each isoform in the presence of pathogen infection. The possible regulation of defense-related enzymes in imparting host resistance is discussed. http://www.phytoparasitica.org posting March 11, 2008.     

Over-expression of the Arabidopsis <Emphasis Type="BoldItalic">NPR1</Emphasis> gene in citrus increases resistance to citrus canker

Citrus canker, caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc), is a serious leaf and fruit spotting disease affecting many important citrus cultivars including grapefruit and certain sweet oranges. Currently, efficacious and economical disease control measures for highly susceptible citrus cultivars are lacking. Development of commercial cultivars with greater field resistance to citrus canker is the optimum strategy for effective disease management. In this study, we generated transgenic ‘Duncan’ grapefruit (DG) and ‘Hamlin’ sweet orange (Ham) expressing the Arabidopsis NPR1 gene (AtNPR1), which is a key positive regulator of the long-lasting broad-spectrum resistance known as systemic acquired resistance (SAR). Our results indicate that over-expression of AtNPR1 in citrus increases resistance to citrus canker and that the resistance is related with the expression levels of AtNPR1 in the transgenic plants. The line (DG 42-2) with the highest expression level of AtNPR1 was also the most resistant, which developed significant fewer lesions accompanied by a ten-fold reduction in Xcc population. The lesions developed on DG 42-2 were smaller and darker than those on the control and lacked callus formation. These lesion phenotypes resemble those on canker resistant kumquats and canker susceptible citrus trees treated with SAR-inducing compounds. Therefore, over-expression of AtNPR1 in citrus is a promising approach for development of more resistant cultivars to citrus canker.     

Bacterial fruit blotch of melon: screens for disease tolerance and role of seed transmission in pathogenicity

Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovorax avenae subsp. citrulli, is a serious threat to the watermelon and melon industries. To date, there are no commercial cultivars of cucurbit crops resistant to the disease. Here we assessed the level of tolerance to bacterial fruit blotch of various commercial cultivars as well as breeding and wild lines of melon, using seed-transmission assays and seedling-inoculation experiments. Selected cultivars were also tested in a greenhouse experiment with mature plants. All tested cultivars/lines were found to be susceptible to the pathogen, and most of them showed different responses (relative tolerance vs. susceptibility) in the different assays; however, some consistent trends were found: cv. ADIR339 was relatively tolerant in all tested assays, and cv. 6407 and wild lines BLB-B and EAD-B were relatively tolerant in seed-transmission assays. We also provide evidence supporting a strong correlation between the level of susceptibility of a cultivar/line and the ability of the pathogen to adhere to or penetrate the seed. To the best of our knowledge, this is the first attempt to assess melon cultivars/lines for bacterial fruit blotch response.     

Genetic Differentiation and Host Specificity Among Populations of Alternaria spp. Causing Brown Spot of Grapefruit and Tangerine x Grapefruit Hybrids in Florida

ABSTRACT Alternaria spp. were sampled from brown spot lesions in several geographically separated citrus groves and different grapefruit and tangerine x grapefruit hybrid cultivars in Florida and screened for variation at 16 putative random amplified polymorphic DNA loci. Populations of the pathogen on two hybrids, Minneola and Orlando, in five locations throughout Florida were moderately differentiated (Nei's coefficient of gene differentiation [G(ST)] = 0.12) among locations. The hypothesis that host-specialized forms of Alternaria spp. cause brown spot on different Citrus spp. and cultivars was tested by estimating genetic differentiation among isolates sampled from different hosts and by pathogenicity assays. Isolates sampled from grapefruit and the hybrid cv. Nova were genetically distinct from isolates sampled from other hybrid cultivars including Robinson, Sunburst, Minneola, Orlando, and Murcott. No differentiation could be detected among isolates sampled from this latter group of hybrids. Quantitative pathogenicity assays on leaves using spray inoculation revealed that 'Nova' isolates were not significantly more pathogenic on 'Nova' compared with isolates from 'Minneola' and 'Orlando'. Similarly, grapefruit isolates were not significantly more pathogenic on grapefruit compared with isolates from 'Minneola'. Isolates from all hosts had similar disease rankings on each inoculated cultivar, with 'Minneola' the most susceptible, followed in decreasing order of susceptibility by 'Orlando', 'Sunburst', 'Nova', and 'Duncan' grapefruit. Rough lemon was generally immune to all isolates tested; however, occasional brown spot lesions were observed on leaves of this host with isolates from grapefruit. No evidence was found to support the hypothesis that unique genotypes of the pathogen, which are more virulent on 'Sunburst' or grapefruit, have been introduced to Florida. Populations of Alternaria spp. causing brown spot of citrus on grapefruit and 'Nova' in Florida are genetically distinct from isolates on other cultivars, and we speculate that these populations are in the early stages of adaptation to and possible speciation on these hosts.     

Impact of carrot resistance on development of the <Emphasis Type="Italic">Alternaria</Emphasis> leaf blight pathogen (<Emphasis Type="Italic">Alternaria dauci</Emphasis>)

The interaction between Alternaria dauci and two carrot cultivars differing in their resistance to leaf blight was investigated by microscopy. The fungal development between 1 and 15 days post-inoculation was quite similar in the susceptible cv. Presto and the partially resistant cv. Texto: After conidial germination, leaf adhesion of the pathogen was achieved with mucilaginous filaments; hyphae penetrated the leaves directly with/without the formation of appressoria-like structures or via stomata; the fungus spread by epiphytic hyphae with hyphopodia and subcuticular mycelia. Intense necrotic plant cell reactions occurred under the fungal structures. At 21 days post-inoculation, typical features of fungal development were noted for each cultivar: growing hyphae emerged from stomata in cv. Presto, whereas conidiophores without conidia were observed in cv. Texto. Leaf tissues of both cultivars were strongly damaged and vesicle-like structures (assumed to be plant phenolics) were abundantly present between mesophyll cells. A real-time PCR method was developed for in planta quantification of A. dauci. Between 1 and 15 days post-inoculation, the fungal biomass was equivalent in the two cultivars and was about fourfold higher in cv. Presto than cv. Texto at 21 and 25 days post-inoculation. Taken together, our results indicated that A. dauci was able to colonize both cultivars in a similar manner during the first steps of the interaction, then fungal development in the partially resistant cultivar was restricted due to putative plant defence reactions. The results of this study enhance the overall understanding of infection processes in the A. dauci-carrot pathosystem.     

Survey of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis> (CTV) diversity in pigmented <Emphasis Type="Italic">Citrus x paradisi</Emphasis> (Macfad.) (Grapefruit) trees in north-western Argentina

Citrus tristeza virus (CTV) is the most severe viral pathogen of citrus and elicits a wide range of devastating disease symptoms. Grapefruit cultivars (Citrus x paradisi) are the most sensitive among citrus to the effects of CTV infections. Grapefruit is an important crop within the north-western Argentine citrus industry; however, production has been affected by CTV stem-pitting. In general, CTV diversity within South America is poorly studied, with data on grapefruit CTV populations being particularly limited. In this study, 50 samples were collected from Star Ruby, Henninger’s Ruby and Ruben Pink cultivars, within the provinces of Tucumán, Salta and Jujuy in north-western Argentina. The CTV p33 gene was PCR amplified and the resulting amplicons sequenced with Sanger sequencing. A subset of these amplicons was sequenced with Illumina MiSeq sequencing. AT-1-like sequences were dominant within the majority of populations, as determined by Sanger sequencing, followed by sequences clustering within the unresolved Kpg3/SP/T3 and RB clades. Sequencing by Illumina MiSeq confirmed this, as well as detecting minor sequence types within the HA 16–5, VT, B165 and A18 clades.     

木虱传播柑桔黄龙病及电镜检验报告

 本试验在福建省福州市及广东省侥平县两地进行。1978年以来,在福州市以蕉柑、福桔和新会甜橙病树为毒源树,以柑桔实生健苗为指示植物,进行木虱传试验。结果在329株指示苗中,发病40株,发病率12.2%,潜育期短的半年,长的5年。对照指示苗72株均未见发病。1982~1984年,在饶平县以蕉柑病树为毒源树,以椪柑实生健苗为指示植物,共试验70株,发病33株,发病率47.1%,潜育期短的2个月,长的8个月。对照指示苗9株未见发病。木虱传染病苗的病状与毒源树的病状相同,均表现黄梢和叶片斑驳。获"毒"木虱的唾液腺和脂肪体的超薄切片样本在电镜下均观察到原核细胞微生物(称为类立次克体或类细菌)。唾液腺内的病原体分布在外皮层细胞,内皮层细胞和腺腔内。其菌体的形状大小和膜壁结构等特征均与毒源树及木虱传染病苗的筛管细胞内的相同,木虱获得和传递柑桔黄龙病病原的方式与蚜虫传播病毒中的持久性类型十分相似。     

A comparative assessment of potential components of partial disease resistance to <Emphasis Type="Italic">Fusarium</Emphasis> head blight using a detached leaf assay of wheat,barley and oats

The relative resistance of 15 winter barley, three winter wheat and three winter oat cultivars on the UK recommended list 2003 and two spring wheat cultivars on the Irish 2003 recommended list were evaluated using Microdochium nivale in detached leaf assays to further understand components of partial disease resistance (PDR) and Fusarium head blight (FHB) resistance across cereal species. Barley cultivars showed incubation periods comparable to, and latent periods longer than the most FHB resistant Irish and UK wheat cultivars evaluated. In addition, lesions on barley differed from those on wheat as they were not visibly chlorotic when placed over a light box until sporulation occurred, in contrast to wheat cultivars where chlorosis of the infected area occurred when lesions first developed. The pattern of delayed chlorosis of the infected leaf tissue and longer latent periods indicate that resistances are expressed in barley after the incubation period is observed, and that these temporarily arrest the development of mycelium and sporulation. Incubation periods were longer for oats compared to barley or wheat cultivars. However, oat cultivars differed from both wheat and barley in that mycelial growth was observed before obvious tissue damage was detected under macroscopic examination, indicating tolerance of infection rather than inhibition of pathogen development, and morphology of sporodochia differed, appearing less well developed and being much less abundant. Longer latent periods have previously been related to greater FHB resistance in wheat. The present results suggest the longer latent periods of barley and oat cultivars, than wheat, are likely to play a role in overall FHB resistance if under the same genetic control as PDR components expressed in the head. However the limited range of incubation and latent periods observed within barley and oat cultivars evaluated was in contrast with wheat where incubation and latent periods were shorter and more variable among genotypes. The significance of the various combinations of PDR components detected in the detached leaf assay as components of FHB resistance in each crop requires further investigation, particularly with regard to the apparent tolerance of infection in oats and necrosis in barley, after the incubation period is observed, associated with retardation of mycelial growth and sporulation.