小麦新抗源一粒葡抗条锈病的组织学和超微结构研究

 采用荧光显微镜、微分干涉显微镜和电子显微镜技术,系统研究了小麦新抗源一粒葡抗小麦条锈病的组织学和超微结构特征。结果表明:相对于感病品种铭贤169,一粒葡对条锈菌的侵染,在组织学和超微结构上均表现出明显的抗性特征。在组织学水平,表现为菌丝生长受抑,菌落发育延迟或败育,吸器母细胞和吸器数目明显减少;同时,侵染点的寄主细胞表现出不同程度的过敏性坏死症状。电镜观察发现,在一粒葡和感病品种中,条锈菌均可由芽管顶端直接进入或通过形成附着胞进入小麦气孔。其后,在一粒葡上,病菌胞间菌丝、吸器母细胞、吸器在细胞和亚细胞水平均发生了一系列异常变化,表现为原生质染色逐渐加深,液泡增多变大,逐渐消解原生质;胞间菌丝、吸器母细胞细胞壁不规则增厚;胞间菌丝线粒体肿胀,数目增多,逐渐解体;吸器母细胞细胞质逐渐空泡化后丧失其生理功能;吸器外质膜皱褶;吸器外间质加宽并有丝状或颗粒状物质形成,吸器体壁逐渐消解出现孔洞,吸器体最终畸形坏死。同时,寄主细胞产生一系列显著的结构防卫反应:形成胞壁沉积物、乳突、吸器鞘等结构,以及发生坏死,阻碍并抑制病菌的发育及扩展。     

Scanning electron microscopy study of infection structure formation of Puccinia graminis f.sp. tritici in host and non-host cereal species

The development of uredospore-derived infection structures of Puccinia graminis f.sp. tritici in wheat, barley, sorghum and maize was examined by scanning electron microscopy (SEM). Germ tubes grew over the leaf surface until a stoma was located. An appressorium formed over the stoma and the leaf was penetrated by an infection peg. Within the substomatal chamber of all species the infection peg developed a substomatal vesicle by 6 h post-inoculation (hpi). from which a primary infection hypha developed parallel to the long axis of the leaf. In wheat, barley and maize, when a primary infection hypha abutted onto a host cell, a septum was laid down between the tip of the hypha and the substomatal vesicle, delimiting a haustorial mother cell by 12 hpi; haustorial mother cells did not form in sorghum. Secondary infection hyphae arose on the substomatal vesicle side of the septum; infection did not progress further in maize, but in wheat and barley secondary infection hyphae branched, and proliferated intercellularly forming the fungal thallus. A haustorial mother cell was delimited when an intercellular hypha abutted onto a host cell. Infection sites with haustorial mother cells were observed at 12 hpi in barley and 24 hpi in wheat. In all four plant species, some atypical substomatal vesicle initials, substomatal vesicles and primary infection hyphae were observed.     

小麦叶锈菌在感病寄主上发育的组织病理学和超微结构研究

 应用荧光显微技术、微分干涉技术和生物电镜技术,系统地研究了小麦叶锈菌在感病寄主上的发育过程及其超微结构特征。小麦叶锈菌在感病品种上的发育过程可分为几个明显的阶段,即孢子的萌发、附着胞的形成、气孔下囊的分化、初生菌丝和次生菌丝的形成和生长、吸器母细胞和吸器的形成、夏孢子床和夏孢子堆的产生以及夏孢子的形成。小麦叶锈菌的胞间菌丝呈丝状,生长和分枝通常沿寄主细胞壁进行。胞间菌丝与寄主细胞的接触诱导了吸器母细胞的分化,吸器母细胞在与寄主细胞壁的接触部位发育形成入侵栓,穿透寄主细胞壁后于细胞内形成吸器。胞间菌丝和吸器母细胞均含有双核,而成熟吸器则含有单核。经常规染色后,胞间菌丝和吸器母细胞的壁与隔膜均可分辨出由多层构成。     

Comparative microscopic and molecular analysis of Thatcher near‐isogenic lines with wheat leaf rust resistance genes Lr2a,Lr3, LrB or Lr9 upon challenge with different Puccinia triticina races

Thatcher near‐isogenic lines (NILs) of wheat carrying resistance gene Lr2a, Lr3, LrB or Lr9 were inoculated with Puccinia triticina races of virulence phenotype BBBD, MBDS, SBDG and FBDJ. Puccinia triticina infection structures were analysed under the fluorescence microscope over a course of 14 days after inoculation (dai). The relative proportion of P. triticina and wheat genomic DNA in infected leaves was estimated with a semiquantitative multiplex PCR analysis using P. triticina‐ and wheat‐specific primers. The occurrence of a hypersensitive response (HR), cellular lignification and callose deposition in inoculated plants was investigated microscopically. In interactions producing highly resistant infection type (IT) ‘0;’, a maximum of two haustorial mother cells per infection site were produced, and there was no increase in the proportion of P.  triticina genomic DNA in infected leaves, indicating the absence of P. triticina growth. In comparison, sizes of P. triticina colonies increased gradually in interactions producing moderately resistant IT ‘1’ and ‘2’, with the highest proportion of P. triticina genomic DNA found in leaves sampled at 14 dai. In interactions producing susceptible IT ‘3–4’, the highest proportion of P. triticina genomic DNA was found in leaves sampled at 10 dai (45·5–51·5%). HR and cellular lignification were induced in interactions producing IT ‘0;’ and ‘1’ at 1 dai but they were not observed in interactions producing IT ‘2’ until 2 dai. No HR or cellular lignification were induced in interactions producing susceptible IT ‘3–4’. Furthermore, a strong deposition of callose was induced in Lr9 + BBBD and Lr9 + FBDJ (IT ‘0;’), whereas this defence response was not induced in resistant or susceptible interactions involving Lr2a, Lr3 or LrB, indicating that Lr9 mediated resistance was different from that conditioned by Lr2a, Lr3 or LrB.     

菜豆锈病菌侵染对寄主超微结构的作用及菜豆抗锈病的细胞学表现

 在电镜下观察发现,菜豆锈病菌侵染菜豆后,逐步对其超微结构产生影响:寄主细胞发生质壁分离;叶绿体变形;叶绿体的片层结构排列零乱;线粒体脊模糊不清,直至叶绿体解体;线粒体空泡化;少数细胞的细胞壁分解;不同细胞的细胞器堆积在一起。同时,病原菌的侵染激发了寄主抗病性的细胞学表现:供试的抗感菜豆品种都表现为在病原菌侵入位点的寄主细胞壁内侧有高电子致密物质沉积;与吸器母细胞接触的寄主细胞壁加厚以及在吸器颈周围有电子不透明物质形成。只是这3种反应在抗病品种中表现得更加强烈。此外,抗病品种中还有一些特有的抗性特征,如被侵染细胞及其相邻细胞的快速坏死,吸器母细胞侵入位点的寄主细胞壁外侧也有一种高电子致密物质沉积,抗病品种中真菌吸器周围聚集含大量线粒体的寄主细胞的细胞质,且吸器外基质比感病品种中的宽。     

小麦与条锈病菌不亲和互作的超微结构

 采用条锈菌同一小种的野生型菌系和弱毒突变菌系,分别接种同一小麦品种的方法,研究了不亲和互作的超微结构特征。在不亲和互作中,条锈菌的胞间菌丝、吸器母细胞和吸器明显受抑。吸器可以在发育早期受抑坏死,也可迟滞至吸器体形成之后坏死。吸器外质膜严重皱褶,并出现孔洞,吸器外间质加宽,沉积大量电子致密物质。侵染位点的小麦叶肉细胞表现与过敏性坏死反应相关联的一系列变化。细胞壁内侧还出现乳突状或颗粒状沉积物等防御结构或物质。     

种衣剂17号包衣对小麦条锈菌影响的组织病理学研究

种衣剂１７号内含三唑醇等杀菌剂和其它化学成分，小麦种子用种衣剂包衣，出苗后一叶期接咱小麦条锈菌毒性小种，采用荧光染色技术和整叶透明技术，研究了种衣剂１７号对条锈菌在寄主内发育的影响。观察结果表明，该种衣剂对对叶表的夏孢子萌发和芽管入侵无明显影响，但可使菌丝扩展和菌丝分枝严重受抑，吸器母细胞和吸器形成的数目减少，每一侵染点的吸器一般不超过２个，并且对吸器具有致畸作用。在多数侵染点内可观察到寄主细胞坏     

116个小麦品种（系）抗叶锈基因Lr9-Lr26、Lr19-Lr20的复合PCR检测

[目的]建立简单、快速、有效的小麦抗叶锈基因复合PCR体系,从而提高分子标记辅助选择效率。[方法]以28个‘Thatcher’为背景的近等基因系和16个已知基因载体品系作为试材,测试了小麦抗叶锈病基因Lr9、Lr26、Lr19和Lr20的STS标记特异性,通过优化PCR反应体系和循环条件,构建了抗叶锈基因Lr9-Lr26和Lr19-Lr20的复合PCR检测体系。对116个小麦品种(系)所含有的抗叶锈病基因进行了分子检测。[结果]供试品种均不含有Lr9和Lr20,47个品种含有Lr26(基因频率为40.5%),‘中梁22’含有Lr19。经反复验证,Lr9-Lr26和Lr19-Lr20复合PCR技术检测结果可靠,且与上述单个分子标记检测结果一致。[结论]建立的Lr9-Lr26和Lr19-Lr20的复合PCR检测体系可以准确、稳定、高效地检测小麦抗叶锈基因Lr9、Lr26、Lr19和Lr20。     

High and low pre-inoculation temperatures decrease the effectiveness of the Lr 20 and Sr 15 rust resistance genes in wheat

Spring wheat seedlings containing Lr 20 and Sr 15 resistance alleles were raised at 30° C, prior to inoculation with leaf rust ( Puccinia recondita race 76–2,3) and stem rust ( Puccinia graminis f.sp, tritici race 343–1,2,3,5,6) pathogens, respectively. Infected plants were then grown at one of seven temperatures in the range 18–30 C and infection types were scored at 10 days post-inoculation. These results were compared with those obtained for plants raised at a pre-inoculation temperature of 18° C. In both 18° C and 30° C pre-grown plants, a progressive increase in infection type was observed on resistant lines as post-inoculation temperature increased. However, resistant lines raised at 30°C had significantly higher infection types than plants raised at 18° C at all post-inoculation temperatures for which some degree of resistance was still evident in the plants raised at 18°C, The maximum temperature for expression of resistance was significantly higher for Lr 20 than for Sr 15. irrespective of pre-inoculation temperature. A lowering of the resistance expression was also evident in Sr 15 -bearing lines raised at a very low pre-inoculation temperature (4°C). The effects of low pre-inoculation temperature on resistance were assessed in both winter and spring wheat lines. These results are discussed in the light of current ideas concerning the host membrane location of pathogen recognition events.     

Ultrastructural and Cytochemical Investigations of the Antagonistic Effect of Verticillium lecanii on Cucumber Powdery Mildew

ABSTRACT Chronological events of the intercellular interaction between Verticillium lecanii and cucumber powdery mildew, caused by Sphaerotheca fuliginea, were investigated at different times after inoculation by transmission electron microscopy. V. lecanii hyphae colonized host structures by tight binding, apparently mediated by a thin mucilaginous matrix. As early as 24 h after application of the antagonist, increased vacuolation and disorganization of the cytoplasm of the pathogen hyphae were easily detected. By 36 h after treatment, plasmalemma retraction and local cytoplasm aggregation were typical features of damage. Labeling chitin with the wheat germ agglutinin (WGA)/ovomucoid-gold complex showed that intracellular invasion of S. fuliginea by V. lecanii did not cause extensive host cell wall alterations, except in the area of hyphal penetration. By 48 h after inoculation, further cytoplasm disorganization was observed, as evidenced by the loss of cell turgor and contortion of the cell wall. Such deformation suggests that penetration of the antagonist results from mechanical pressure or localized enzymatic hydrolysis through the action of chitinases, as confirmed by the pattern of labeling obtained with the WGA/ovomucoid-gold complex. By 72 h after contact between the fungi, S. fuliginea cells were markedly collapsed, depleted of their protoplasm due to extensive multiplication of the antagonist, and totally encircled by the antagonist. Based on the current observations, the antagonism of S. fuliginea by V. lecanii appears to involve the following events: (i) attachment of the antagonist to the powdery mildew fungus; (ii) mechanical pressure and production of cell-wall degrading enzymes such as chitinases; (iii) penetration and active growth of the antagonist inside the pathogen hyphae; and (iv) digestion of host tissues and release of the antagonist from dead cells of S. fuliginea. The interaction between V. lecanii and S. fuliginea also affected the morphological and structural features of the haustorial bodies, as shown by increased vacuolation, distortion, and necrotization of the haustorial lobes. These observations provide the first experimental evidence that V. lecanii, primarily known as an entomopathogenic fungus, also has the potential to colonize mycelial structures of S. fuliginea. V. lecanii, therefore, may become a valuable alternative to current management of cucumber powdery mildew in greenhouses.     

小麦-条锈菌互作过程中活性氧及保护酶系的变化研究

 对条锈菌与小麦不同互作体系中组织学特征、活性氧产生及其相关酶系的变化进行了研究。组织学观察表明:非亲和组合相对于亲和组合存在明显差异,表现为菌丝生长受抑,吸器母细胞和吸器形成减少,寄主细胞在接种后18h左右出现过敏性坏死。生化测定结果表明:在非亲和组合中,O₂^-的产生速率和H₂O₂的含量均高于亲和组合,且O₂^-产生速率在接种后12h达到一个峰值,H₂O₂的含量在接种后20和72h出现2个高峰。而在亲和组合中,O₂^-产生速率低于对照或与对照相似,H₂O₂的含量虽然高于对照,但却普遍低于非亲和组合;SOD在亲和组合中的活性总体上要高于非亲和组合;接种24h后,CAT在2种组合中的活性均高于对照,在接种后36和48h时,亲和组合中的CAT活性高于非亲和组合,而在接种60h后又开始低于非亲和组合;POD活性在接种24h后均明显升高,但亲和组合中POD活性增幅大;MDA在非亲和组合中于接种后72h含量明显上升。结果表明,亲和与非亲和组合中条锈菌扩展、活性氧的产生及相关酶活变化都存在明显差异,这些差异与小麦抗锈性的表达可能有密切联系。     

Changes in phenylalanine ammonia-lyase and peroxidase activities in wheat cultivars expressing resistance to the leaf-rust fungus

Phenylalanine ammonia-lyase (PAL) and peroxidase activities increased slightly during pre-haustorial stages of development of the leaf-rust fungus ( Puccinia recondita f. sp. tritici ) in each combination of rust strain and wheat line used. Activity of both enzymes greatly increased during the formation of the first haustoria by avirulent and virulent strains in an Lr28-bearing line but not in an Lr20 -bearing line. PAL increased markedly in activity at the time when an avirulent strain was known to cause hypersensitivity in the Lr28 -bearing line and when another avirulent strain was known to cause cellular changes that preceded the expression of resistance in the Lr20 -bearing line. PAL increased less markedly and not at all at corresponding times when virulent strains developed in the Lr28 and Lr20 lines, respectively. Increases in peroxidase activity occurred after the increases in PAL activity and were greater during resistance expression than in leaves infected with virulent strains. Cytochemical tests revealed enhancements of peroxidase activity in epidermal and guard cells in response to avirulent and virulent strains and in mesophyll cells that were becoming necrotic in response to avirulent strains only. The implications of the changes are that aromatic and phenylpropanoid synthesis and peroxidations may be enhanced in wheat by infection and particularly during resistance expression. The products of these processes may play roles in both Lr28- and Lr20 -based expression of resistance.     

Histological and ultrastructural comparisons of compatible, incompatible and -β-amino- n -butyric acid-induced resistance responses of pepper stems to Phytophthora capsici

The compatible interaction of pepper stems with Phytophthora capsici showed more rapid and severe disease development than did the incompatible interaction, although pathogen penetration styles of host cells in compatible and incompatible interactions were similar to each other. Treatment with -β-amino- n -butyric acid (BABA) protected the pepper plants against P. capsici infection. Reduced hyphal growth and sporangial formation were found after P. capsici infection in BABA-induced resistant and incompatible reactions. One of the most noticeable ultrastructural features of the BABA-induced resistant reaction was the formation of electron-dense wall appositions. The thick and dense wall appositions that encased the haustoria restricted haustorial development, thus leading to limitation of further pathogen penetration into inner plant tissues. A main host response in the incompatible interaction was the occlusion of cortical cells with an amorphous material. Plugging of the intercellular spaces in the cortical cells with electron opaque material was frequently observed in the incompatible interaction, but not in the compatible interaction. Another common feature of the BABA-induced resistant and incompatible reactions was degeneration of mitochondrial structure within penetrating hyphal cytoplasm. The mitochondrial structure in the BABA-induced resistant or incompatible reactions had no distinct double membrane layer and well-shaped cristae.     

多堆柄锈菌侵染玉米的细胞学及超微结构特征

为明确玉米对多堆柄锈菌Puccinia polysora侵染后病理反应的细胞学特征,利用扫描和透射电镜技术分析了玉米自交系与多堆柄锈菌互作中二者的细胞变化过程。多堆柄锈菌对玉米的侵染主要以直接穿透叶片表皮侵入为主,少量可从气孔和细胞间隙侵入。接种后,病菌夏孢子在感病自交系叶片上快速并大量萌发,在叶表生长蔓延并侵入表皮组织细胞,7 d后形成夏孢子堆;在抗病自交系上,病菌萌发、菌丝生长均受到明显抑制,少量入侵的病菌也由于寄主细胞死亡而导致菌丝和夏孢子干瘪死亡。侵染早期在感病寄主细胞间隙出现菌丝并穿透细胞壁,在胞内产生分枝菌丝,此时寄主细胞结构正常;随着菌丝进一步扩展,叶绿体等结构发生紊乱,被侵染细胞逐渐死亡。在抗病自交系上,接菌24 h后寄主即出现过敏性坏死反应,侵入位点与周围细胞快速坏死,抑制菌丝生长蔓延;叶绿体中清晰可见深色颗粒状物质;72 h后细胞壁外侧产生大量致密的深色结晶体,应为与抗病反应相关的酚类物质。表明抗多堆柄锈菌的玉米材料可能存在2种抗病途径,即寄主与病菌互作中由分子识别引起的免疫反应和病菌侵入后的系统防卫反应。     

Histochemical and chemical evidence for lignin accumulation during the expression of resistance to leaf rust fungi in wheat

Levels of individual phenolic acids were examined in primary leaves of wheat (Triticum aestivum) after inoculation with avirulent and virulent strains of the leaf rust fungus (Puccinia recondita f. sp. tritici) at stages when previous work had shown fungal and host cells to be affected by expression of the Lr20 or Lr28 alleles for resistance. The predominant phenolic acid, ferulic acid, as well as p-coumaric and syringic acids were detected in primary leaves in both unbound and bound forms. They were not detected in germinating urediniospores of either rust strain. Levels of unbound phenolic acids changed little in response to infection. In Lr28-bearing leaves inoculated with an avirulent strain, increased concentrations of bound phenolic acids and three other unidentified compounds were observed about 4 h after many single or small groups of cells had undergone hypersensitive collapse. In an Lr20-bearing cultivar, levels of bound phenolic acids fell in leaves inoculated with either a virulent or avirulent rust strain. Coniferyl aldehyde and coniferyl alcohol were not detected in healthy or inoculated leaves of either wheat cultivar. Attempts to affect expression of resistance by application of inhibitors of phenylalanine ammonia-lyase were not successful and both wheat cultivars remained resistant to avirulent rust strains. The bound phenolic acids which accumulate in cells undergoing a hypersensitive response may play a role in resistance of Lr28-bearing wheat to the leaf rust fungus.     

Charakterisierung der Virulenz einer Braunrost-Feldpopulation (Puccinia triticina f. sp. tritici) und Nachweis effektiver Braun-rost-resistenz-gene in Weizen (Triticum aestivum)

Puccinia triticina f. sp. tritici, the causal agent of leaf rust causes yield losses in wheat up to 60%. In order to avoid such losses, leaf rust resistance (Lr-) genes have been incorporated into wheat cultivars. The Lr- genes confer mostly vertical resistance, i.e. they are race specific. Therefore, knowledge of still effective resistance genes is required for efficient breeding of resistant cultivars. To get information on these virulences, a leaf rust population was monitored in field experiments in 2010. For this purpose naturally infection at three different timepoints of wheat development was monitored on Thatcher near isogenic lines (NILs) carrying 37 and accessions carrying 6 additional Lr-genes. Thatcher-NILs carrying Lr2a, Lr9, Lr19, Lr22a, Lr23, Lr24, Lr35, Lr38 and Lr49 showed a significantly lower infection with Puccinia triticina than the susceptible cultivar Thatcher. Thatcher-NILs carrying Lr13, Lr16, Lr37 and Lr46 showed no significant differences in comparison to Thatcher. In order to get information on the effectiveness of resistance genes, P. triticina isolates were collected from the NILs analysed in field trials and a leaf segment test was conducted followed by microscopic analyses. In the field and in the leaf segment test Lr9, Lr19, Lr24 and Lr38 and to some extent Lr3a turned out to be the most effective genes. By microscopic analyses, the infection process as well defense reactions activated before macroscopic symptoms are visible were monitored. By counting haustorial mother cells, it could be demonstrated which Lr-genes provide resistance, which were overcame and whether P. triticina isolates exist already at a low frequency, which may overcome a certain Lr-gene in the future. Thus microscopy offers a timesaving and effective method to detect susceptible or resistant plants and the upcoming of virulent races prior to typical symptom expression.     

Effects of tebuconazole on morphology,structure, cell wall components and trichothecene production of Fusarium culmorum in vitro

The effects of tebuconazole, a systemic fungicide, on the morphology, structure, cell wall components and toxin production of Fusarium culmorum were investigated in vitro. Treatment was by application of four filter paper strips (0.75 cm × 5.0 cm) soaked in 20 µg ml ^?1 fungicide placed around a point inoculum in Petri dishes. Mycelial growth was strongly inhibited by fungicide treatment. Scanning electron microscopic observations showed that the fungicide caused irregular swelling and excessive branching of hyphae. The morphological changes induced by the fungicide at the ultrastructural level included considerable thickening of the hyphal cell walls, excessive septation, the formation of the incomplete septa, extensive vacuolisation, accumulation of lipid bodies and progressing necrosis or degeneration of the hyphal cytoplasm. Non‐membrane inclusion bodies were often detected in the hyphal cytoplasm. Furthermore, the formation of new hyphae (daughter hyphae) inside collapsed hyphal cells was common following treatment. The daughter hyphae also displayed severe alterations such as irregular thickening of the cell walls and necrosis of the cytoplasm. Using cytochemical techniques, the labelling densities of chitin and β‐1,3‐glucan in the cell walls of the fungicide‐treated hyphae were more pronounced than in those of the control hyphae. Moreover, immunogold labelling with antiserum against deoxynivalenol (DON) revealed that Fusarium toxin DON was localized in the cell walls, cytoplasm, mitochondria and vacuoles of the hyphae from the control and the fungicide treatment, but the labelling density in the fungicide‐treated hyphae decreased dramatically compared with the control hyphae, indicating that tebuconazole reduced Fusarium toxin production of the fungus. © 2001 Society of Chemical Industry     

The establishment of systemic infection in leaves of oilseed rape by Leptosphaeria maculans

In a series of growth room experiments in which leaves of Brassica napus var. oleifera were inoculated with ascospores or pycnidiospores of Leptosphaeria maculans successful infections progressed through three consecutive phases. Initial establishment in the mesophyll was succeeded by a phase of intercellular exploration, when hyphal proliferation was highly variable and host cell necrosis always ensued, and then by a systemic phase when hyphae were consistently sparse. Host cells associated with the hyphal front were capable of autofluorescence, accumulation of vital stains and plasmolysis, indicating that they were viable and that the pathogen was biotrophic throughout this sequence. During either of the first two phases permanent fungistatic containment, involving the formation of vesicles by disintegration of the hyphae, often occurred. Localization at the first phase was symptomless; at the second it was signified by a lesion with a clearly defined margin.
There was a negative correlation between biotrophic potential and necrotrophic potential of three pathogenic isolates, on both the moderately susceptible cultivar Primor and the resistant cultivar Jet Neuf. As leaves aged, a progressively larger proportion of infections failed to become systemic. With increasing inoculum load, symptomless localization of infection diminished, the phase of necrosis extended, and the probability of irreversible systemic development increased.     

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.