Ultrastructural Characterization of Infection and Colonization of Maize Leaves by Colletotrichum graminicola, and by a C. graminicola Pathogenicity Mutant

ABSTRACT Observations were made of the ultrastructure of infection and colonization of leaves of a susceptible maize inbred by Colletotrichum graminicola and by a C. graminicola pathogenicity mutant. The mutant causes no symptoms on either maize leaves or stalks. Prior evidence suggested that it is deficient in production of signal peptidase, responsible for cleavage of signal peptides from proteins destined for transport through the endoplasmic reticulum. There was no significant difference in the process of infection or colonization by the mutant and wild-type strains up to 48 h after inoculation. Both the mutant and the wild type produced globose, melanized appressoria within 24 h after inoculation on the host surface. By 36 h, both strains had penetrated the host epidermal cells directly. The host cells frequently formed papillae in response to appressoria, but these were not usually successful in preventing fungal ingress in either case. Penetration was followed by formation of irregularly shaped, swollen infection hyphae. Infection hyphae of both strains grew biotrophically for a relatively short time (less than 12 h). One or more hyphal branches was produced from each infection hypha, and these invaded adjacent mesophyll cells. Both strains of the fungus grew cell-to-cell, setting up new biotrophic interactions in each cell, between 36 and 48 h after inoculation. Papillae were frequently formed by the mesophyll cells, but these were not successful in preventing fungal ingress. The first noticeable difference between the mutant and the wild type was related to their interaction with mesophyll cells. Cells invaded by the wild type died relatively quickly, whereas those infected by the mutant appeared to survive longer. The most dramatic difference between the mutant and wild type occurred when the mutant completely failed to make a transition to necrotrophic growth, while the wild type made that switch at 48 to 72 h after inoculation. The mutant may be unable to secrete sufficient quantities of one or more proteins that are necessary to support the switch between biotrophy and necrotrophy.     

Hypersensitive cell death and post-haustorial defence responses arrest the orange rust (Hemileia vastatrix) growth in resistant coffee leaves

The growth of a coffee orange rust fungus (Hemileia vastatrix Berk and Br.) isolate (race II) and the sequence of responses it induced in leaves of resistant Coffea arabica L. and C. congensis Froehner as well as on a susceptible C. arabica were investigated cytologically and biochemically. The percentages of germinated urediospores and of appressoria formed over stomata as well as the fungal growth inside leaf tissues were similar in resistant and susceptible leaves until the 3rd day after the inoculation. In the susceptible leaves, at the majority of the infection sites (70%) the fungus pursued its growth without apparent inhibition while in the resistant leaves the fungus ceased its growth with higher frequency (34% in C. arabica and 54% in C. congensis) after the formation of at least one haustorium. The first signs of incompatibility, detected 2 days after the inoculation, were cytologically expressed by hypersensitive host cell death (HR), host cell wall autofluorescence and haustoria encasement with callose and β-1,4-glucans. Biochemically, two peaks of phenylalanine ammonia-lyase (PAL) activity were detected by 2 and 5 days after the inoculation. The 1st peak coincided with the early accumulation of phenolic compounds and with the beginning of cell death. The 2nd peak could be related to later accumulation of phenols and the lignification of the host cell walls. About 5–7 days after the inoculation, ultrastructural observations revealed the accumulation of a material partially crystallized in the intercellular spaces around the senescent hyphae, next to dead host cells and in close association with the middle lamella that initially labelled for pectins. It also contained polysaccharides and phenolic-like compounds. Cellulose, hemicellulose, extensins, hydroxyproline-rich glycoproteins and proteins were not detected. The hypertrophy of the host cells in the infection area were also observed around 12 days after the inoculation corresponding macroscopically to the reaction flt.In susceptible plants, cell death was also observed 3 days after the inoculation but only in a reduced percentage of infection sites in which the fungus aborted at an early stage. A late haustorium encasement and stimulation of PAL activity were also observed but these delayed host responses did not prevent fungal growth and sporulation.The intercellular material, only observed in the resistant plants, is here reported for the first time and although its role is unknown it might be the result of plant cell death.     

Effect of foliar diseases of wheat on the physiological processes affecting yield under semi-arid conditions

The effects of Septoria leaf blotch (caused by Mycosphaerella graminicola) and leaf rust (caused by Puccinia recondita ) on the physiological processes affecting yield were studied under semi-arid conditions in Israel. In the absence of water stress, photosynthetic activity decreased as the rate of disease development increased. Transpirational activity was reduced in leaves infected by Septoria leaf blotch, but intensified in leaves with slight leaf rust infections (0.5-5% severity). When leaf rust seventy exceeded 5%, the transpirational water loss declined. Both diseases were associated with a decrease in the maximum rate of grain-weight accumulation, but did not affect the duration of weight accumulation. An inverse linear relationship between disease and final grain weight was observed. Under conditions of water stress, photosynthetic activity of leaves slightly infected by Septoria leaf blotch (1-5"., severity) was greater than that of uninfected leaves. Severely infected leaves (" 25% severity) photosynthesized the least. Both diseases were associated with a decrease in the maximum rate of grain weight accumulation, but the duration of accumulation was longer in tillers with a slow rate of disease development than in uninfected tillers. The relationship between disease and final grain weight in these cases was parabolic; yield was increased at low levels of infection. Theoretical explanations and practical implications of these phenomena are presented and discussed.     

The effect of dose and mobility on the strength of selection for DMI fungicide resistance in inoculated field experiments

Plots of spring wheat cv. Baldus were inoculated at GS 13 with four Mycosphaerella graminicola isolates, two relatively susceptible and two relatively resistant to DMI fungicides. Changes in the ratio of relatively susceptible to resistant types following fungicide or water sprays were measured. Three fungicides were compared: flutriafol, which is very mobile within leaves, fluquinconazole, which is less so, and prochloraz, which is almost immobile. All are inhibitors of sterol demethylation. In 1996, fungicide-treated plots were sprayed once with half the recommended dose at GS 39–47. In 1997, three doses were used: one-quarter and one-eighth of the recommended dose and a dual application of two one-eighth recommended doses, a week apart. Isolates were classified using a discriminating dose assay and the ratio of relatively susceptible to relatively resistant isolates in each field plot before and after fungicide application calculated. In both years, the numbers of relatively susceptible and relatively resistant isolates were equal just before fungicide application. All fungicides caused significant selection towards resistance, but the strength of selection varied with fungicide, dose and position in the crop canopy. Fluquinconazole selected most strongly and gave the best control of disease. Interactions between fungicide and dose were not significant. Selection was equally strong all along leaves sprayed with prochloraz, but increased smoothly from base to tip of leaves sprayed with fluquinconazole or flutriafol. Averaged over fungicides, reducing the dose of a single fungicide application from one-quarter to one-eighth slightly reduced selection towards resistance on both leaf layers. The dual one-eighth dose caused twice the change of the single one-eighth dose on the flag leaf, but was similar to a single spray on leaf 2.     

Colonization of Fiber Cells by Colletotrichum graminicola in Wounded Maize Stalks

ABSTRACT Colonization of wounded maize stalks by a wild-type strain of Colletotrichum graminicola was compared with colonization by a C. graminicola mutant that is avirulent on maize leaves, and by a wild-type strain of C. sublineolum that is normally a pathogen of sorghum but not maize. Local infection by all strains at the wound site resulted in formation of primary lesions consisting of disintegrated parenchyma cells beneath an intact rind and epidermis. However, subsequent rapid longitudinal expansion of the primary lesion occurred only in infections with the wild-type C. graminicola strain, and proceeded specifically through the fiber cells associated with the vascular bundles and the rind. Hyphae emerged from the fiber cells to produce discontinuous secondary lesions. There was no evidence that C. graminicola is a vascular wilt pathogen. Resistance of wounded cv. Jubilee maize stalks to the mutant strain of C. graminicola and to C. sublineolum was associated with restriction of colonization and spread of the pathogen through the fibers, as well as with the limitation of localized destruction of parenchyma cells at the wound site.     

Effect of Figaren, an Antiviral Protein from Cucumis figarei, on Cucumber mosaic virus Infection

Local lesion formation on cowpea leaves was more than 50% inhibited by treatment with a 23 kDa RNase-like glycoprotein from Cucumis figarei, figaren, from 24 hr before to 1 hr after inoculation with Cucumber mosaic virus (CMV). CMV accumulation detected by ELISA in tobacco leaves treated with figaren 6 or 0 hr before inoculation with CMV was suppressed. When upper leaves of tobacco plants were treated with figaren and inoculated 10 min later with CMV, mosaic symptoms were delayed for 5–7 days on most of the tobacco plants, and some plants remained asymptomatic. From fluorescence in situ hybridization, infection sites were present in figaren-treated cowpea or melon leaves after inoculation with CMV, though the sites were reduced in number and size compared with those in water-treated control leaves. The amount of CMV RNAs and CMV antigen in melon protoplasts inoculated with CMV and subsequently incubated with figaren similarly increased with time as did that in the control. ELISA and local lesion assays indicated that CMV infection on the upper surfaces of the leaves of tobacco, melon, cowpea and C. amaranticolor whose lower surfaces had been treated with figaren 5–10 min before CMV inoculation was almost completely inhibited. Figaren did not inhibit CMV infection on the opposite untreated leaf halves of melon, cowpea and C. amaranticolor, whereas it almost completely inhibited CMV infection on the untreated halves of leaves of tobacco. CMV infection was not inhibited in the untreated upper or lower leaves of the four plants. These data suggest that figaren does not completely prevent CMV invasion but does inhibit the initial infection processes. It may also induce localized acquired resistance in host plants. Received 10 October 2000/ Accepted in revised form 6 February 2001     

生物拮抗菌Trichoderma harzianum处理玉米种子诱导实生苗的抗病性分析

 本文研究了生物拮抗菌Trichoderma harzianum(T22)处理对玉米(Mo17)实生苗生长的影响,以及对病原菌Colletrichonum graminicola的抗病性。试验结果表明:用拮抗菌T.harzianum处理后能明显地提高玉米苗根系发育和叶片生长,显著的诱导玉米叶片和根系中防御相关的酶活性和蛋白含量。用病原菌C.graminicola接种玉米叶片后观测发现,处理的实生苗叶片上病斑直径明显小于不处理的对照。尽管拮抗菌T.harzianum诱导植物抗病性的作用已被证实,但该研究更进一步揭示了T.harzianum处理玉米种子后植株的抗性应答与促进实生苗根系发育和叶片生长,以及诱导抗性相关的酶。     

Wheat Leaf Rust Uredospore Production on Adult Plants: Influence of Leaf Nitrogen Content and Septoria tritici Blotch

ABSTRACT Leaf rust uredospore production and lesion size were measured on flag leaves of adult wheat plants in a glasshouse for different lesion densities. We estimated the spore weight produced per square centimeter of infected leaf, per lesion, and per unit of sporulating area. Three levels of fertilization were applied to the plants to obtain different nitrogen content for the inoculated leaves. In a fourth treatment, we evaluated the effect of Septoria tritici blotch on leaf rust uredospore production. The nitrogen and carbon content of the spores was unaffected or marginally affected by lesion density, host leaf nitrogen content, or the presence of Mycosphaerella graminicola on the same leaf. In leaves with a low-nitrogen content, spore production per lesion was reduced, but lesion size was unaffected. A threshold effect of leaf nitrogen content in spore production was however, evident, since production was similar in the medium- and high-fertilizer treatments. In leaves inoculated with M. graminicola and Puccinia triticina, the rust lesions were smaller and produced fewer spores. The relationships among rust lesion density, lesion size, and uredospore production were fitted to a model. We determined that the density effect on spore production resulted mainly from a reduction in lesion size, the spore production per unit of sporulating surface being largely independent of lesion density. These results are consistent with those obtained previously on wheat seedlings. The main difference was that the sporulation period lasted longer in adult leaves.     

高温处理西花蓟马若虫对其雌成虫寿命、繁殖力及后代发育的影响

温度是影响昆虫生长发育的重要生态因素之一,高温逆境作为一种绿色防控手段可以有效控制温室害虫种群发展。为了探究高温处理西花蓟马若虫对其种群发展的影响,本试验用41℃和43℃两个温度分别处理西花蓟马初孵若虫2、6、12、24和36h后,观察并记录其雌成虫寿命、繁殖力及后代发育指标的变化情况。结果表明,随着处理时间的延长,两个高温处理后西花蓟马雌成虫寿命,子代若虫总数、成虫总数和总存活率(若虫发育到成虫的存活率)均显著下降,后代雌雄性比呈总体下降趋势。41℃处理后,性比从2.30∶1降低到2.13∶1;43℃处理后从2.25∶1降低到2.07∶1,均明显低于对照的2.69∶1。另外,两性生殖种群相比于孤雌生殖种群更易遭受高温处理的影响。     

Hydroxyproline-rich glycoprotein accumulation in tobacco leaves protected against Erysiphe cichoracearum by potato virus Y infection

Tobacco cv. Havana 425 acquired resistance to a compatible isolate of Erysiphe cichoracearum after infection by a strain of potato virus Y (PVY^N) that causes veinal necrosis; another common strain (PVY^O) that does not cause necrosis gave less protection. Hydroxyproline-rich glycoproteins (HRGPs), believed to be involved in resistance, were determined by analysing hydroxyproline (Hyp) in purified cell walls. Hyp content increased significantly in PVY^N-protected leaves, compared with untreated controls, 2–4 days after necrotic lesion symptoms developed. No further increase in Hyp was noted in PVY^N-protected leaves after E. cichoracearum challenge. Hyp increases were significantly higher in protected leaves of plants showing symptoms on day 7 than on day 10. Infection with the PVY^O strain caused significant decrease in Hyp content, compared with uninoculated controls. Inoculation of virus-free plants with E. cichoracearum induced moderate and transitory Hyp increases on day 2 or 3, followed by a quick decrease associated with a weak response by the compatible host. It is suggested that HRGP accumulation induced by PVY^N (but not by PVY^O) causes changes in the host cell wall that result in resistance to E. cichoracearum .     

Subcuticular-Intracellular Hemibiotrophic and Intercellular Necrotrophic Development of Colletotrichum acutatum on Almond

ABSTRACT The early infection and colonization processes of Colletotrichum acutatum on leaves and petals of two almond cultivars with different susceptibility to anthracnose (i.e., cvs. Carmel and Nonpareil) were examined using digital image analysis of light micrographs and histological techniques. Inoculated tissue surfaces were evaluated at selected times after inoculation and incubation at 20 degrees C. Depth maps and line profiles of the digital image analysis allowed rapid depth quantification of fungal colonization in numerous tissue samples. The results showed that the early development of C. acutatum on petals was different from that on leaf tissue. On petals, conidia germinated more rapidly, germ tubes were longer, and fewer appressoria developed than on leaves. On both tissues, penetration by the pathogen occurred from appressoria and host colonization was first subcuticular and then intracellular. On petals, colonizing hyphae were first observed 24 h after inoculation and incubation at 20 degrees C, whereas on leaves they were seen 48 to 72 h after inoculation. Intercellular hyphae were formed before host cells became necrotic and macroscopic lesions developed on petals >/=48 h and on leaves >/=96 h after inoculation. Histological studies complemented data obtained by digital image analysis and showed that the fungus produced infection vesicles and broad hyphae below the cuticle and in epidermal cells. In both tissues, during the first 24 to 48 h after penetration fungal colonization was biotrophic based on the presence of healthy host cells adjacent to fungal hyphae. Later, during intercellular growth, the host-pathogen interaction became necrotrophic with collapsed host cells. Quantitative differences in appressorium formation and host colonization were found between the two almond cultivars studied. Thus, on the less susceptible cv. Nonpareil fewer appressoria developed and host colonization was reduced compared with that on cv. Carmel.     

Formation of phytoalexins within and outside lesions of Botrytis cinerea in French bean leaves

Rapidly spreading lesions and lesions restricted in size developed in primary leaves of French bean (Phaseolus vulgaris) in response to infection byBotrytis cinera isolates BC-1 and BC-5, respectively. These isolates caused similar differential lesions in leaves of cucumber, flax, lettuce and tomato. To determine whether phytoalexin accumulation was correlated with the resistant reaction in bean leaves, accumulation of phytoalexins was examined in necrotic areas of both types of lesions and in their surrounding green tissues. Phaseollin was the predominant phytoalexin, both inside and outside lesions, whereas phaseollidin and sometimes also phaseollinisoflavan were always present in lower concentrations. Phaseollin accumulated earlier and to higher levels within and around lesions of isolate BC-5 than of isolate BC-1. Relatively low concentrations of phaseollin were detected in the more remote green areas, including the petiole, of leaves bearing a spreading lesion. The phaseollin metabolite, 6a-hydroxyphaseollin, was found only inside lesions and in a narrow zone around lesions of both types. The authors consider the possibility that the differing concentrations of phytoalexins in the infected tissues are not a determining factor for the differential interactions betweenB. cinerea and bean leaves, but are rather the result of it.     

根结线虫在中国的一新纪录种——拟禾本科根结线虫Meloidogyne graminicol

本文描述了采自海南省三亚市葱 ( Allium tistulosum)上的拟禾本科根结线虫 Meloidogynegraminicola。该种雌虫的会阴花纹多为上下长的卵圆形 ;线纹细弱 ,通常平滑连续 ,但在局部区域骤然出现将线纹截断的短线纹 ;侧区不明显 ;侧尾腺口小 ,相隔很近。雄虫的头冠与头区等宽 ,头区很低、光滑或有 2条不明显的环纹。二龄幼虫的尾相当长 ,尾透明末端的界限通常明显 ,尾端细长 ,末端呈细棍棒状。拟禾本科根结线虫在中国属首次报道 ,其寄主葱及其所属的百合科为拟禾本科根结线虫寄主植物的新纪录种和新纪录科     

Frequency of Sexual Reproduction by Mycosphaerella graminicola on Partially Resistant Wheat Cultivars

ABSTRACT The frequency of sexual reproduction has a profound effect on the population structure and the adaptive potential of a facultatively sexual parasite. Little is known about the relationship of quantitative host resistance to the frequency of sex in pathogens. We sampled over 5,000 fungal fruiting bodies from eight different wheat cultivars over a 3-year period. The cultivars possessed varying degrees of susceptibility to Mycosphaerella graminicola, a facultatively sexual pathogen that is hetero-thallic and bipolar. The fruiting bodies were classified as M. graminicola pycnidia or ascocarps (asexual and sexual fruiting bodies, respectively), other identifiable fungi, or unidentified. In all 3 years, area under the disease progress curve (AUDPC) explained a significant proportion of the variation in ascocarps as a percentage of M. graminicola fruiting bodies (P < 0.0005). The mean percentage of M. graminicola ascocarps from all cultivars was 63% in 1998, when the epidemic was intense, and 14% in 1999, a year of low disease levels. In 2000, samples were taken at 7-day intervals from 6 June to 27 June from two cultivars with substantially different AUDPCs (788 and 2,185 percentage-days). The less diseased cultivar yielded its first M. graminicola ascocarps 1 week later than the more diseased cultivar, and respective means of ascocarps as a percentage of M. graminicola fruiting bodies across sampling dates were 20.2 and 59.3%. The ratio of sexual to asexual reproduction by M. graminicola is likely to be strongly conditioned by infection density.     

Quantitative PCR monitoring of the effect of azoxystrobin treatments on Mycosphaerella graminicola epidemics in the field

Quantitative PCR and visual monitoring of Mycosphaerella graminicola epidemics were performed to investigate the effect of curative and preventative applications of azoxystrobin in wheat field crops. A non-systemic protectant and a systemic curative fungicide, chlorothalonil and epoxiconazole, respectively, were used as references. PCR diagnosis detected leaf infection by M graminicola 3 weeks before symptom appearance, thereby allowing a clear distinction between curative and preventative treatments. When applied 1 week after the beginning of infection, azoxystrobin curative activity was intermediate between chlorothalonil (low effect) and epoxiconazole. When applied preventatively, none of the fungicides completely prevented leaf infection. There was some indication that azoxystrobin preventative treatments may delay fungal DNA increase more than epoxiconazole at the beginning of leaf infection. Both curative and preventative treatments increased the time lapse between the earliest PCR detection and the measurement of a 10% necrotic leaf area. Azoxystrobin only slightly decreased the speed of necrotic area increase compared with epoxiconazole. Hence, azoxystrobin activity toward M graminicola mainly resides in lengthening the time lapse between the earliest PCR detection and the measurement of a 10% necrotic leaf area. Information generated in this way is useful for optimal positioning of azoxystrobin treatments on M graminicola.     

Effects of temperature, leaf wetness and cultivar on the latent period of Mycosphaerella graminicola on winter wheat

After inoculation of winter wheat cv. Longbow at a single time, lesions of M. graminicola were produced over a long interval starting 15–35 days after inoculation, dependent on temperature. There was no evidence that a single infection gave rise to more than one lesion. After the initial infection period at 100% relative humidity (r.h.), keeping leaves wet for c. 10 h per day did not shorten latent period on seedlings. Experiments in controlled-environment chambers demonstrated a minimum latent period at approximately 17°C Variation in the latent period of individual lesions was also minimum at this temperature. The latent period varied among the cultivars tested, cv. Longbow having the shortest, cv. Avalon having almost the longest. Field observations broadly confirmed the results of experiments in constant-environment chambers.     

Differential induction of superoxide dismutase in downy mildew-resistant and -susceptible genotypes of pearl millet

Differential induction of superoxide dismutase (SOD) in downy mildew-resistant and -susceptible genotypes of pearl millet ( Pennisetum glaucum ) was observed on inoculation with Sclerospora graminicola . SOD activity was studied in resistant (IP18292) and susceptible (23B) pearl millet seedlings inoculated with S. graminicola . SOD activity increased by 2·3-fold in resistant seedlings upon inoculation. SOD activity was greatest in roots, with a specific activity of 3182 U per mg protein, after inoculation. SOD activity increased in all the resistant genotypes upon inoculation with S. graminicola . Native PAGE analysis showed four isozymes of SOD, three of which (SOD-1, -2 and -4) were Cu/Zn-SOD, whereas isozyme SOD-3 was Mn-SOD. This study also revealed increased intensity of all four isozymes of SOD in the resistant genotype upon inoculation. The involvement of SOD in pearl millet (host)–downy mildew pathogen interaction is discussed.     

Infection of the pearl millet (Pennisetum americanum) inflorescence by the downy mildew fungus (Sclerospora graminicola)

Different developmental stages of the inflorescence of pearl millet (Pennisetum americanum) were inoculated with zoospores of the downy mildew fungus (Sclerospora graminicola). Individual florets within a panicle were infected, with resultant malformation of any floral organs that were not fully differentiated at the time of infection. 'Green-ear' symptoms resulting from hyperplasia and hypertrophy of the host tissues were accompanied by both sexual and asexual sporulation of the fungus on the malformed plant parts. No grain set occurred in affected florets, indicating that secondary inoculum was able to cause yield reductions even at late stages in the host development. Infection of differentiated stigmas led to rapid dissolution and necrosis of tissue and prevented colonization by the pathogen. This failure suggests that seeds are unlikely to be infected internally.     

(+)-catechin acts as an infection-inhibiting factor in strawberry leaf

ABSTRACT An infection-inhibiting factor (IIF) was isolated from strawberry leaves and identified as (+)-catechin. This compound inhibited the formation of infection hyphae from appressoria of Alternaria alternata, but allowed both spore germination and appressorial formation. It is a normal component of strawberry leaves, but further accumulates as the major IIF in response to inoculation with nonpathogenic spores of A. alternata. The accumulation of (+)-catechin on a susceptible host was not induced, however, by inoculation with pathogenic spores of the strawberry pathotype or by inoculation with nonpathogenic spores supplemented with host-specific toxin (AF-toxin I). These results imply that (+)-catechin acts as a protective agent during induced resistance and that AF-toxin I acts as a fungal suppressor of induced resistance.