Histopathology of compatibility and incompatibility between oilseed rape and Albugo Candida

Cotyledons of one resistant and three susceptible rape lines/cultivars were inoculated with zoospores of Albugo Candida race 7. Samples of whole cotyledons were examined by differential interference contrast microscopy. The time course of the infection process was followed histologically. Germination of zoospore cysts occurred 2-3 h after inoculation. Infection was initiated with germ-tubes penetrating through stomata. Haustorium formation was first observed in the palisade mesophyll cells adjacent to the substomatal chambers 8 h after inoculation.
Only after the establishment of the first haustorium did compatible and incompatible interactions begin to differentiate. In the resistant cultivar, most primary hyphae produced single haustoria. Necrosis of the invaded host cell was first observed 12 h after inoculation followed by cessation of fungal growth. The death of host cells was largely restricted to the penetration site; the adjacent non-penetrated cells remained apparently unaffected. In the susceptible hosts, necrosis of infected cells occurred only infrequently, and hyphal growth continued unabated, resulting in mycelial ramification into the mesophyll. Numerous haustoria were produced.
Histological studies showed that the earliest event distinguishing a compatible from an incompatible interaction occurred after formation of the first haustorium and that resistance was not manifested until the host mesophyll cell had come into contact with the first haustorium. The distinction between compatibility and incompatibility was substantiated by quantitative analysis of white rust development on both resistant and susceptible lines/cultivars.     

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.     

大豆疫霉菌对大豆下胚轴侵染过程的细胞学研究

 接种后1.5~24h,用光镜和电镜研究了2个大豆品种与大豆疫霉菌Ps411的亲和性和非亲和性互作。观察结果表明,大豆疫霉菌对大豆下胚轴的侵染过程可分为侵入前、侵入、皮层组织中的扩展和进入维管束组织4个连续阶段。大豆下胚轴接种后在25℃保湿培养,1.5h后游动孢子即形成休止孢并萌发产生附着孢,3h后侵入表皮细胞,6h后进入皮层组织,24h后进入维管束组织。病原菌主要以侵染菌丝直接侵入表皮,表皮细胞间隙是主要侵入部位。皮层细胞是病原菌定殖和发展的主要场所,胞间菌丝侵入皮层细胞并形成吸器。在菌丝与寄主细胞接触部位的寄主细胞壁与质膜之间常有胞壁沉积物的形成。在抗病品种上病菌的侵染事件与感病品种基本一致,但不能形成正常的吸器,胞壁沉积物明显多于感病品种,菌丝在寄主组织内的扩展明显受到抑制。利用β-1,3-葡聚糖免疫金标记单克隆抗体进行的免疫细胞化学的研究表明,胞壁沉积物内含有大量的β-1,3-葡聚糖,在大豆疫霉菌菌丝壁中也存在β-1,3-葡聚糖。以上结果表明,病原菌的侵染可诱导抗病寄主细胞内β-1,3-葡聚糖迅速的合成与积累、并形成胞壁沉积物,以抵御病菌的侵染与扩展。     

A biochemical mechanism for the gene-for-gene resistance of tomato to Cladosporium fulvum

Model experiments were carried out with the tomato varieties Moneymaker (no resistance genes), Leaf Mould Resister No. 1 (resistance gene Cf 1), Vetomold (resistance gene Cf 2) and V 473 (resistance genes Cf 1 and Cf 2) and various physiological races ofCladosporium fulvum. Leaking of³²P from labelled leaf disks, was obtained on infiltration with high molecular weight excretion products from incompatible races ofC. fulvum but not with those from compatible races. These products were obtained by Sephadex G-25 gel filtration of culture filtrates.The observations are in line with our hypothesis that the gene-for-gene relation existing between tomato andC. fulvum is based on interaction of specific fungal excretion products with specific receptors in the host which may be located in the cell membrane. The presence of these fungal compounds is supposed to be controlled by four avirulence genes (A₁, A₂, A₃ and A₄) and that of the receptors by the four resistance genes (Cf 1, Cf 2, Cf 3 and Cf 4). Results obtained from experiments with tomatoes Cf 1, Cf 2 and Cf 1 Cf 2 suggest that leakage followed by the hypersensitivity reaction occurs whenC. fulvum races possessing a specific avirulence allele penetrate into a host carrying the corresponding resistance allele.It is not yet clear why growth ofC. fulvum is stopped when leakage of the host tissue resulting in the hypersensitive reaction takes place. No compound toxic toC. fulvum is present or is formed in homogenates of tomato leaves.Samenvatting Modelproeven werden uitgevoerd met de tomatenvariëteiten Moneymaker (geen resistentiegenen), Leaf Mould Resister No. 1 (resistentiegen Cf 1), Vetomold (resistentiegen Cf 2), V 473 (resistentiegen Cf 1 en Cf 2) en verschillende fysiologischen rassen vanCladosporium fulvum. Bladponsjes van radioactief gemerkte bladeren (gemerkt met³²P) werden geïnfiltreerd en geïncubeerd met cultuurfiltraatfracties (fractionering over Sephadex G-25) vanC.fluvum. Waargenomen werd dat ponsjes, behandeld met cultuurfiltraat van een niet compatibeleC.fulvum, een grotere uitlek van radioactief gemerkt materiaal te zien gaven dan in de, gevallen waarin een compatibele schimmel werd gebruikt.Deze waarnemingen stemden overeen met onze hypothese dat de gen-om-gen relatie die bestaat tussen tomaten enC.fulvum, gebaseerd, is op een interactie van specifieke schimmelprodukten met specifieke receptoren in de plantencellen, mogelijk in de membranen. De produktie van de specifieke stoffen door de schimmel zou worden bepaald door vier avirulentiegenen (A₁, A₂, A₃ en A₄), en de aanwezigheid van de specifieke receptoren in de plantencel door de vier resistentiegenen Cf 1, Cf 2, Cf 3 en Cf 4.De waarnemingen, verkregen uit de proeven met de tomaten Cf 1, Cf 2 en Cf 1 Cf 2, doen vermoeden dat de uitlek een gevolg is van een overgevoeligheidsreactie die optreedt, indien een fysio vanC.fulvum, dat een specifiek avirulentie allel bezit, een gastheer binnendringt die beschikt over een bijpassend resistentie allel.Het is tot nu toe niet duidelijk waarom de groei vanC.fulvum stopt indien, uitlek op gaat treden als gevolg van de overgevoeligheidsreactie. In homogenaten van tomatenbladeren werd geen stof gevonden die de groei vanC.fulvum remt.     

Accumulation of pathogenesis-related proteins in the epidermis of tomato leaves infected by Cladosporium fulvum

Upon infection byCladosporium fulvum, tomato plants start to produce pathogenesis-related (PR) proteins. The PR proteins 1,3-β-glucanase, chitinase, and PR-1b accumulated near the stomata in the lower epidermis ofC. fulvum-inoculated tomato leaves as could be determined by immunolocalization with polyclonal antibodies. However, no differences in accumulation of PR proteins between a compatible and an incompatible interaction were found. Results obtained from enzyme activity measurements of 1,3-β-glucanase and chitinase on similar leaf material as used for the immunolocalization did not fully reflect the immunolocalization data. The antibodies possibly detect only the extracellular but not the intracellular enzymes. The accumulation of PR proteins near the stomata might be part of a general defence response of plants against pathogens and potential pathogens.     

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.     

Characterization of elicitor activities of apoplastic fluids isolated from tomato lines infected with new races of Cladosporium fulvum

Apoplastic fluids were isolated from the near-isogenic lines Cf2, Cf4 and Cf5 of the tomato (Lycopersicon esculentum Mill.) cultivar ‘Moneymaker’ infected with a set of newCladosporium fulvum races 2.5, 2.4.11, 2.5.9, 2.4.5.11, 2.4.9.11 and 2.4.5.9.11 and a set of previously described races 2, 4, 5, 2.4., 2.4.5 and 2.4.5.9. Upon injection of the apoplastic fluids into leaves of the near-isogenic lines Cf0, Cf2, Cf4, Cf5 and Cf9 and the genotypes carrying Cf6 and Cf11 chlorosis or necrosis was observed in all plants that were resistant to the race that initially had been used to raise the apoplastic fluid, except in the genotype Cf11 when the races 4, 2.4, 2.4.5 and 2.4.5.9 from our own collection were involved. The latter races may contain virulence gene all as our collection had not yet been checked for presence of this virulence gene. Apoplastic fluids of all races induced chlorosis on the genotype carrying resistance gene Cf6, a gene which has not yet been overcome by any of the presently described races ofC. fulvum.Low pH non-denaturing polyacrylamide gel electrophoresis showed that apoplastic fluids of compatible interactions with the new races 2.5.9, 2.4.9.11 and 2.4.5.9.11 carrying the virulence gene a9, like the previously described race 2.4.5.9, did not contain the necrosis-inducing peptide, while the peptide was present in all races carrying avirulence gene A9. This indicates that this race-specific elicitor is strongly associated with avirulence gene A9 and is possibly its product as has been suggested previously.     

Differences in disc gel electrophorese pattern of soluble proteins excreted by different physiological races and isolates of Cladosporium fulvum

The extracellular soluble proteins of 15 different isolates comprising 9 different physiological races ofCladosporium fulvum were harvested from 3 to 4 weeks old shake cultures. The culture filtrates were purified by gel filtration over Sephadex G-25 and the V_o-fractions were freeze-dried. These preparations were submitted to disc gel electrophoresis on polyacrylamide gels at pH 9.5 at circa 4°C. The electrophoretical protein patterns appeared to differ for each isolate irrespective of the physiological race to which they belong. The various isolates originated from different sites in the Netherlands.     

稻瘟病菌在水稻CO39近等基因系上的生命表和繁殖表

 本文选用以CO39为背景的水稻近等基因系与广东省稻瘟病菌3个优势生理小种(ZC₁₃、ZB₁和ZB₅)构成12个亲和性和非亲和性互作组合,组建各组合稻瘟病菌的生命表和繁殖表,分析在亲和性互作中稻瘟病菌的生存和繁殖状况对寄主发病的影响,并探讨在非亲和性互作中抗性基因在抵御病菌侵染过程中的作用。结果表明,在亲和性和非亲和性互作中,稻瘟病菌生长繁殖各阶段均参与了感抗反应并发挥了一定的作用:在CO39近等基因系上,同一小种的孢子萌发率或菌丝形成率无明显差别,而小种间则表现一定差异;病菌的定殖和繁殖是亲和与非亲和互作的关键阶段,定殖率、病斑形成率低,则寄主表现抗病,反之则表现感病。在繁殖表众多表示种群增长的参数中,世代平均产孢量F'、净增殖率R₀、最大相对增长率r_max和种群趋势指数Ⅰ等4个指标适合于反映稻瘟病菌繁殖状况与感抗病反应的关系。     

Studies on the Infection Process of Fusarium Culmorum in Wheat Spikes: Degradation of Host Cell Wall Components and Localization of Trichothecene Toxins in Infected Tissue

After single spikelet inoculation, the infection process of Fusarium culmorum and spread of fungal hyphae in the spike tissues were studied by scanning and transmission electron microscopy. While hyphal growth on outer surfaces of the spike was scanty and no successful penetration was observed, the fungus developed a dense mycelium on the inner surfaces and effectively invaded the lemma, glume, palea and ovary by penetration pegs. During the inter- and intracellular spreading of the fungus, marked alterations in the host tissues were observed, including degeneration of cytoplasm, cell organelles, and depositions of electron dense material between cell wall and plasmalemma. Ultrastructural studies revealed that host cell walls in proximity of the penetration peg and in contact with hyphae were less dense or transparent which suggested that cell wall degrading enzymes were involved in colonisation of host tissues by fungal hyphae. Enzyme- and immunogold-labelling investigations confirmed involvement of extracellular enzymes, that is cellulases, xylanases and pectinases, in degradation of cell wall components. Localization studies of trichothecenes indicated that toxins could be detected in host tissues at an early stage of infection.     

Infection by Magnaporthe oryzae chrysovirus 1 strain A triggers reduced virulence and pathogenic race conversion of its host fungus, <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) is associated with an impaired growth phenotype of its host fungus, Magnaporthe oryzae. In this report, we assayed the virulence and pathogenicity of MoCV1-A-infected and MoCV1-A-free M. oryzae on rice plants. MoCV1-A infection did not affect virulence-associated fungal traits, such as conidial germination and appressorium formation. However, after punch inoculation of leaves on rice plants, MoCV1-A-infected strain formed smaller lesions than the MoCV1-A-free strain did on all rice varieties tested, showing that MoCV1-A infection resulted in reduced virulence of host fungi in rice plants. In contrast, after spray inoculation of rice seedlings, in some cases, MoCV1-A-infected and MoCV1-A-free strains caused different lesion types (resistance to susceptible, or vice versa) on individual international differential rice varieties. However, we did not find any gain/loss of the fungal avirulence genes by PCR, suggesting that MoCV1-A infection can convert the pathogenicity of the host M. oryzae from avirulence to virulence, or from virulence to avirulence, depending on the rice variety. We also confirmed the correlation of these race conversion events and invasive hyphae growth of the fungi in a leaf sheath inoculation assay. These data suggested that MoCV1-A infection generally confers hypovirulence to the fungal host and could be a driving force to generate physiological diversity, including pathogenic races.     

Carbohydrate composition of apoplastic fluids isolated from tomato leaves inoculated with virulent or avirulent races of Cladosporium fulvum (syn. Fulvia fulva)

Inoculation of tomato (Lycopersicon esculentum) with virulent races ofCladosporium fulvum (compatible interactions), resulted in substantial changes of the carbohydrate composition of apoplastic fluids isolated from the leaves, during the course of the infection process. In addition to a decrease in the concentration of the translocation sugar sucrose, a transient accumulation of the hexoses glucose and fructose and an accumulation of the polyol mannitol were observed. The latter coincided with a rising level of mannitol dehydrogenase, an enzyme that reduces fructose to mannitol. Only minor changes were detected in the carbohydrate composition of apoplastic fluids isolated from leaves of uninoculated control plants or plants inoculated with avirulent races ofC. fulvum (incompatible interactions). The fungal metabolite mannitol was not detected in apoplastic fluids isolated from the latter plants.These results suggest that, upon colonization of the intercellular spaces by virulent races ofC. fulvum, apoplastic sucrose is hydrolyzed by a host and/or fungal invertase and the resulting hexoses, glucose and fructose, are converted into mannitol by the fungus. In incompatible tomato-C. fulvum interactions a functional nutritional relationship between plant and fungus is prevented by plant defense responses, which might explain why in these interactions the carbohydrate composition of apoplastic fluids is similar to that of uninoculated control plants.Samenvatting Inoculatie van tomaat (Lycopersicon esculentum) met virulente fysio's vanC. fulvum (compatibele interacties), leidde tot aanzienlijke veranderingen in de koolhydraatsamenstelling van apoplastische vloeistoffen die uit de bladeren werden geïsoleerd in de loop van het infectieproces. Naast een sterke daling van de concentratie van de transportsuiker saccharose, vond er ophoping van de hexoses glucose en fructose en de polyol mannitol plaats. De accumulatie van mannitol ging gepaard met een toename in de activiteit van mannitol dehydrogenase, een enzym dat fructose reduceert tot mannitol. In de koolhydraatsamenstelling van apoplastische vloeistoffen geïsoleerd uit bladeren van niet geïnoculeerde controleplanten, of planten geïnoculeerd met avirulente fysio's vanC. fulvum (incompatibele interacties), werden slechts kleine veranderingen waargenomen. De schimmelmetaboliet mannitol kon niet worden aangetoond in de apoplastische vloeistoffen die uit deze planten werden geïsoleerd.Deze resultaten suggereren dat bij de kolonisatie van de intercellulaire ruimtes door virulente fysio's vanC. fulvum, saccharose uit de apoplast wordt gehydrolyseerd door invertase afkomstig van de plant of de schimmel waarna de ontstane hexoses, glucose en fructose, door de schimmel worden omgezet in mannitol. Bij incompatibele tomaatC. fulvum interacties wordt een functionele voedingsrelatie tussen plant en schimmel voorkomen door het optreden van afweerreacties van de plant, hetgeen kan verklaren waarom in deze interacties de koolhydraatsamenstelling van apoplastische vloeistoffen vergelijkbaar is met die van niet geïnoculeerde controleplanten.     

Early defence responses induced by AVR9 and mutant analogues in tobacco cell suspensions expressing the Cf-9 resistance gene

The interaction between the fungal leaf pathogen Cladosporium fulvum and its only host, tomato, fits the gene-for-gene model. In tomato, the Cf-9 resistance gene product mediates specific recognition of the fungal avirulence gene product AVR9, resulting in a hypersensitive response and resistance. Cf9 tomato leaves respond with necrosis after injection with AVR9, whereas Cf9 tomato cell suspensions do not show defence responses after treatment with AVR9. Here we report on early defence responses induced in Cf-9 transgenic tobacco leaves and Cf-9 transgenic tobacco cell suspensions after treatment with synthesized AVR9 and mutant analogues R08K, F10A and F21A. The necrosis-inducing activity of the AVR9 peptides increased in the order F21A, F10A, AVR9, R08K. An oxidative burst was induced at a much lower AVR9 peptide concentration as compared to medium alkalization and necrosis. Interestingly, the mutant peptide F21A failed to induce necrosis and medium alkalization but did induce an oxidative burst. In all assays, the relative differential activities of the AVR9 peptides were similar to those observed in Cf9 tomato leaves. Both AVR9 and F21A activated a MAP kinase in Cf-9 transgenic tobacco cell suspensions. AVR9 also induced specific cell death in these suspensions. The relation between the induction of early defence responses and necrosis is discussed.     

Effect of cold‐induced changes in physical and chemical leaf properties on the resistance of winter triticale (×Triticosecale) to the fungal pathogen Microdochium nivale

This study showed that several mechanisms of the basal resistance of winter triticale to Microdochium nivale are cultivar‐dependent and can be induced specifically during plant hardening. Experiments and microscopic observations were conducted on triticale cvs Hewo (able to develop resistance after cold treatment) and Magnat (susceptible to infection despite hardening). In cv. Hewo, cold hardening altered the physical and chemical properties of the leaf surface and prevented both adhesion of M. nivale hyphae to the leaves and direct penetration of the epidermis. Cold‐induced submicron‐ and micron‐scale roughness on the leaf epidermis resulted in superhydrophobicity, restricting fungal adhesion and growth, while the lower permeability and altered chemical composition of the host cell wall protected against tissue digestion by the fungus. The fungal strategy to access the nutrient resources of resistant hosts is the penetration of leaf tissues through stomata, followed by biotrophic intercellular growth of individual hyphae and the formation of haustoria‐like structures within mesophyll cells. In contrast, a destructive necrotrophic fungal lifestyle occurs in susceptible seedlings, despite cold hardening of the plants, with the host epidermis, mesophyll and vascular tissues being digested and becoming disorganized as a result of the low chemical and mechanical stability of the cell wall matrix. This work indicates that specific genetically encoded physical and mechanical properties of the cell wall and leaf tissues that depend on cold hardening are factors that can determine plant resistance against fungal diseases.     

Ultrastructural study on scab resistance expressed in epidermal pectin layers of pear leaves

Proliferation and collapse of subcuticular hyphae of Venturia nashicola race 1 were studied ultrastructurally, after inoculation of susceptible Japanese pear cv. Kousui, resistant Japanese pear cv. Kinchaku, resistant Asian pear strain Mamenashi 12 and nonhost European pear cv. Flemish Beauty leaves, to understand the nature of the resistance mechanism. After cuticle penetration by the pathogen, the hyphae were observed at lower frequency in epidermal pectin layers and middle lamellae of leaves of the three resistant plants than in those of susceptible ones. This result suggested that fungal growth was suppressed in the incompatible interaction between pear and V. nashicola race 1. In the pectin layers of all inoculated plants, some hyphae had modifications such as breaks in the plasmalemma with plasmolysis, necrotic cytoplasm and degraded cell walls. More hyphae had collapsed in the leaves of the three resistant plants than in those of the susceptible cv. Kousui. In collapsed hyphae, the polymerized cell walls broke into numerous fibrous and amorphous pieces, showing that the scab resistance might be associated with cell wall-degrading enzymes from pear plants.     

Histological Aspects of a Hypersensitive Response in Poplar to Melampsora larici-populina

ABSTRACT The course of the infection and development of the biotrophic fungus Melampsora larici-populina on leaf tissue from the hybrid poplar Populus deltoides x P. nigra 'Ogy' was monitored at the histological level. Leaf disks were inoculated with one of two rust physiological races (E1 and E2), resulting in interactions that were either incompatible (race E1) or compatible (race E2). In the compatible interaction, the fungus rapidly colonized the leaf without inducing any apparent host response. Symptoms appeared on the leaf several days after inoculation just prior to spore dissemination. The incompatible interaction was characterized by the early collapse and disorganization of cytoplasm of infected cells 17 h after inoculation and within 2 h after the appearance of the first haustoria. Resistance to M. larici-populina was mediated through a hypersensitive response, since it was extremely localized and involved only the few cells that were in the immediate vicinity of each infected cell.     

Ultrastructural and cytochemical investigations of pathogen development and host reactions in susceptible and partially-resistant carrot roots infected by Pythium violae, the major causal agent for cavity spot

Two carrot genotypes, cultivar Nanco and line 24, susceptible and partially- resistant respectively to cavity spot, were compared ultrastructurally and cytochemically 24 h, 48 h and 72 h after root inoculation with a virulent Pythium violae isolate. The extent of pathogen ingress and the response of the host differed markedly with the two genotypes. In cv Nanco, growth of fungal hyphae was predominantly intracellular and was accompanied by pronounced damage; by 48 h after inoculation, pericycle and the first cell layers of the phloem parenchyma were invaded, resulting in host wall dissolution and cytoplasm aggregation. The growth of P. violae in line 24 was limited to the pericycle, even up to 72 h after inoculation; fungal colonization was accompanied by retraction of cytoplasm and in the appearance of granular or fibrillar material in the host cell lumen. Some affected host cells were filled with structureless osmophilic material. In cultivar Nanco, invading fungal hyphae were unaffected; by contrast in line 24, the cytoplasm of invading hyphae, particularly those inside the cell host, was disorganised and structureless. Infection and host response in the two cultivars were studied with two specific labels: Aplysia gonad lectin (AGL), a polygalacturonic acid-binding lectin, and an exoglucanase complexed to colloidal gold were used to locate pectin and cellulosic -(1,4)-glucans respectively in infected tissues. The decrease of cytochemical labeling beyong fungal penetration showed clearly hydrolysis of pectin and cellulose in cell walls of the cv Nanco. By contrast, the cell wall of line 24 remained largely intact, although, unlabeled amorphous and electron-dense material was observed inside the wall. Fibrillar or electron dense material commonly observed in infected tissue of line 24 apparently did not contain pectic or cellulosic substances. Moreover, material observed in host cells or fungal hyphae was also free of labeling. The origin and the chemical composition of these compounds as well as their possible role in the defence mechanisms of carrot against P. violae are discussed.     

Vascular colonization patterns in susceptible and resistant tomato cultivars inoculated with Fusarium oxysporum f.sp. lycopersici races 0 and 1

The vascular colonization pattern of Fusarium oxysporum f.sp. lycopersici races 0 and 1 in tomato was studied in five susceptible and five resistant cultivar–fungus combinations during a 26-day period after inoculation by root immersion. Propagules spread discontinuously along the stems in all five cultivars 1 day after inoculation, irrespective of cultivar resistance. Five days later the fungus was limited to the stem bases in all cultivars. Between the fifth and 12th days, stem colonization by the fungus stopped in all cultivar–race combinations. Thereafter, the situation remained stable in resistant combinations, with inoculum distributed discontinuously, and no disease symptoms were apparent. By contrast, in the susceptible combinations a gradual upward colonization of the stems was seen such that fungal distribution was no longer discontinuous and disease symptoms appeared. These results suggest that a fungal 'incubation' period in the base of the vascular system is required before a secondary invasion of tissues occurs in susceptible genotypes. The slope of the regression line fitted between the height reached by the fungus up the stem ( y ) and the time after inoculation ( x ) provides a measure of the horizontal (polygenic) resistance in tomato cultivars     

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.