Localized hemibiotrophy in Colletotrichum: cytological and molecular taxonomic similarities among C. destructivum, C. linicola and C. truncatum

The infection process of hemibiotrophic isolates of Colletotrichum linicola (from flax, Linum usitatissimum ) and C . truncatum (from broad bean, Vicia faba and lentil, Lens culinaris ) was studied by light microscopy. Host surfaces were penetrated directly leading to a symptomless, biotrophic phase characterized by the elaboration of large multilobed, multiseptate, vesicular primary hyphae that were restricted to the initially infected epidermal cells. Biotrophy lasted for the first 48 h of the host-pathogen interaction and was rapidly succeeded by a necrotrophic phase during which narrow, secondary hyphae invaded the surrounding leaf tissues and water-soaked spreading lesions with sporulating, monosetate acervuli were produced on infected host surfaces. Molecular taxonomic analysis of the nucleotide sequences of the amplified D2 and ITS-2 regions of rDNA revealed very close similarities (97–99%) between these isolates and those of C . destructivum obtained from cowpea ( Vigna unguiculata ) and lucerne ( Medicago sativa ), and also of C . truncatum obtained from pea ( Pisum sativum ). This association was consistent with results from a comparative assessment of some in-planta and in-vitro morphological and growth characteristics of these hemibiotrophic fungi. It was concluded that localized hemibiotrophy is an infection strategy utilized predominantly by a closely-related group of pathogens comprising C . destructivum , C . linicola and C. truncatum , and the formation of multilobed primary hyphae restricted to the first penetrated cell might therefore be a key taxonomic character which correlates consistently with ITS sequence data.     

Ultrastructure of the Infection of Sorghum bicolor by Colletotrichum sublineolum

ABSTRACT Ultrastructural studies of the infection of susceptible and resistant cultivars of Sorghum bicolor by Colletotrichum sublineolum were conducted. Initial penetration events were the same on both susceptible and resistant cultivars. Germ tubes originating from germinated conidia formed globose, melanized appressoria, that penetrated host epidermal cells directly. Appressoria did not produce appressorial cones, but each penetration pore was surrounded by an annular wall thickening. Inward deformation of the cuticle and localized changes in staining properties of the host cell wall around the infection peg suggests that penetration involves both mechanical force and enzymic dissolution. In compatible interactions, penetration was followed by formation of biotrophic globular infection vesicles in epidermal cells. Filamentous primary hyphae developed from the vesicles and went on to colonize many other host cells as an intracellular mycelium. Host cells initially survived penetration. The host plasma membrane invaginated around infection vesicles and primary hyphae and was appressed tightly to the fungal cell wall, with no detectable matrix layer at the interface. Necrotrophic secondary hyphae appeared after 66 h and ramified through host tissue both intercellularly and intracellularly, forming hypostromatic acervuli by 114 h. Production of secondary hyphae was accompanied by the appearance of electron-opaque material within infected cells. This was thought to represent the host phytoalexin response. In incompatible interactions, infection vesicles and primary hyphae were formed in epidermal cells by 42 h. However, they were encrusted with electron-opaque material and appeared dead. These observations are discussed in relation to the infection processes of other Colletotrichum spp. and the host phytoalexin response.     

Light and scanning electron microscopy studies on the infection process of melon leaves by Colletotrichum lagenarium

Colletotrichum lagenarium is the casual agent of anthracnose disease of melons. Light and scanning electron microscopy were used to observe the infection process of C. lagenarium on the leaves of two melon cultivars differing in susceptibility. On both cultivars conidia began germinating 12 h after inoculation (hai), forming appressoria directly or at the tips of germ-tubes. By 48 hai appressoria had melanised and direct penetration of host tissue had begun. On the susceptible cultivar, infection vesicles formed within 72 hai and developed thick, knotted primary hyphae within epidermal cells. By 96 hai C. lagenarium produced highly branched secondary hyphae that invaded underlying mesophyll cells. After 96 hai, light brown lesions appeared on the leaves, coincident with cell necrosis and invasion by secondary hyphae. While appressoria formed more quickly on the resistant cultivar, fewer germinated to develop biotrophic primary or invasive necrotrophic secondary hyphae than on the susceptible cultivar. These results confirm that C. lagenarium is a hemibiotrophic pathogen, and that resistance in melons restricts colonisation by inhibiting the development of necrotrophic secondary hyphae.     

苹果炭疽叶枯病病原Glomerella cingulata及其侵染过程

为明确苹果炭疽叶枯病病原菌围小丛壳Glomerella cingulata的侵染致病特征,在分离获得该病原菌的基础上,采用形态学观察、ITS序列分析和致病性测定对其进行了鉴定,并利用光学和扫描电子显微镜对病原菌在嘎啦苹果叶片上的侵染过程进行了研究.结果表明,在陕西咸阳地区分离获得的9株病原菌均为围小丛壳G.cingulata.25 ℃下接种9 h后,分生孢子中间产生隔膜,双胞化,并萌发产生芽管和附着胞;24 h后分生孢子的2个细胞均可萌发并形成芽管及附着胞,部分芽管顶端可产生次级分生孢子;48 h后次级分生孢子萌发形成附着胞;72 h后,附着胞下形成的侵染钉可直接入侵寄主,在表皮细胞内形成初生菌丝和次生菌丝,此时叶片表面已出现褐色斑点.接种7 d后叶片病斑处出现分生孢子盘和子囊壳.表明陕西省近年出现的苹果炭疽叶枯病病原菌为围小丛壳G.cingulata,该病菌在嘎啦叶片上的一些特殊侵染行为可能是导致该病害易在短时间内暴发的重要原因.     

柿树炭疽菌侵染柿树叶柄的超微结构观察

 利用透射电镜技术研究了柿树炭疽菌侵染柿树叶柄的超微结构。结果表明:病原菌侵入寄主细胞后,产生细胞内的初生菌丝,其表面沉积凹凸不平的电子不透明物质。一层界面基质(interfacial matrix)把表初生菌丝细胞壁和凹陷的寄主原生质膜分开。随着初生菌丝定殖下一个细胞,原先细胞中的细胞膜消失,形成许多泡囊,随后叶绿体消失,内质网和高尔基体也逐渐降解,最后细胞内物质全部被降解成电子不透明的颗粒,降解的物质沿着初生菌丝和细胞壁表面沉积。初生菌丝穿透细胞壁的过程中,菌丝顶端接触细胞壁后膨大,并在中部产生一个隔膜,然后顶端细胞产生一个较细的穿透菌丝,穿透寄主细胞壁。穿透菌丝在寄主细胞壁中的狭窄处产生一个隔膜,一旦穿透寄主细胞壁后,迅速膨大。次生菌丝在细胞间和细胞内扩展,通过菌丝体对细胞壁施加的机械压力引起寄主细胞壁破裂,或同初生菌丝一起使细胞壁解体。侵染90 h后,形成垫形分生孢子盘。在分生孢子盘周围的表皮细胞中,次生菌丝不断形成子座组织,使原来的子座扩大,子座不断分化形成产梗细胞,产梗细胞产生分生孢子梗,分生孢子梗生长和发育对角质层和表皮细胞壁组织折叠处施加机械压力,使角质层和表皮细胞壁组织进一步折叠,分生孢子盘也相应扩大。     

Infection of Nicotiana species by the anthracnose fungus, Colletotrichum orbiculare

Colletotrichum gloeosporioides f. sp. malvae, isolate Biomal®, ATCC 20767, was originally isolated from round-leaved mallow (Malva pusilla) and developed as a weed biocontrol agent. Ribosomal DNA sequence analysis was recently used to re-classify this fungus as C. orbiculare, which is an aggregate species with a number of formae speciales. Several morphological features of ATCC 20767 were examined that were consistent with those described for C. orbiculare, and inoculation of a number of Nicotiana species and several cultivars of N. tabacum showed that this fungus was pathogenic to many of these previously undescribed hosts. Spore germination and appressorium formation were higher on tobacco than previously observed on round-leaved mallow. The pathogen produced melanized appressoria on N. tabacum leaves that formed preferentially at the anticlinal epidermal cell wall. A symptomless phase of infection persisted for 72–96 h postinoculation, during which time the fungus first produced a spherical infection vesicle from an infection peg, and then large primary hyphae which grew through the epidermal cells. The large primary hyphae were highly constricted at the points of penetration of the host cell walls. Thin secondary hyphae appeared at 96–120 h postinoculation coinciding with the appearance of light green, water-soaked spots and the formation of acervuli. The infection of tobacco by C. orbiculare ATCC 20767 is not a non-specific interaction but appears to follow an intracellular hemibiotrophic infection process that is very similar to that established for the C. orbiculare infection of round-leaved mallow, cucurbits and beans.     

The development of different pathotype groups of Mycosphaerella pinocles in susceptible and partially resistant pea leaves

The progress of infection by high- and low-virulence isolates of Mycosphaerella pinodes was examined in susceptible and partially resistant pea leaves. Conidia germinated with one or more germ tubes which frequently branched and formed appressorium-like structures on the leaf surface. Penetration occurred through the epidermal walls. A structure similar to an infection vesicle was formed, lying partly in the epidermal wail and partly in the cell lumen. From this structure, a penetration hypha was derived which initiated the development of the intra- and intercellularly-growing fungal colony. Infections led to rapid tissue collapse in both susceptible and resistant interactions. In resistant interactions, the formation of infection vesicles and penetration hyphae was reduced, and the development and spread of lesions was retarded.     

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.     

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。     

Ultrastructural study on interaction between a sterile,white endophytic fungus and eggplant roots

Eggplant roots colonized by a sterile, white mycelial endophyte (SWM) were previously found to become highly resistant to Verticillium wilt. SWM alone, however, caused no visible, disease symptoms, such as wilting or necrosis. The mechanism of the symptomless infection by SWM was investigated in this study. Electron microscopy revealed that hyphae of SWM were abundant on and inside the root epidermal cells 2 weeks after inoculation. Many terminal appressoria formed from apical tips of hyphae, and heavy degradation of the host cell walls was evident where hyphae accumulated. By 4 weeks following inoculation, penetration pegs easily breached epidermal cells, and the infection hyphae penetrated outer cortical cells. In response to the hyphal ingress, numerous tubule-like vesicles and membrane-bound, multivesicular bodies accumulated in cortical cytoplasm near the infection sites of the outer cortical cells, but no visible signs of the host reactions were seen in the epidermal cells. Papillae developed at the spaces between cell walls and plasma membranes at the infection sites. The penetration hyphae often grew out of the papillae, but further hyphal ingress was halted in the middle cortical cell layer. By 8 weeks following inoculation, papillae that developed in these cells contained larger amounts of highly electron-dense material and were reinforced by multilamellate, fibrous elements. Hyphae that entered such papillae were confined to them, and the hyphal cytoplasm degenerated. As the result of the activated resistance reactions, root vascular cylinders remained intact, and the host plants did not wilt.     

Scanning electron microscopy of early infection in the uredial stage of Puccinia sorghi in Zea mays

An SEM study was made of the infection process of Puccinia sorghi in Zea mays. A uredospore germ tube grows across epidermal cells and along their anticlinal walls, often branching and altering direction of growth. The fungus, on attaining a stoma, delimits an appressorium over it. Infection peg initials enlarge linearly and centripetally along the appressorium base, forcing open the stomatal slit. Having penetrated the stomatal aperture, the infection peg develops a substomatal vesicle. From the vesicle, two short primary infection hyphae develop synchronously, a septum later forming between the vesicle body and each hyphal base. A further septum divides the primary hypha into two cells. Secondary infection hyphae emerge later from the fully expanded vesicle on the proximal side of each vesicle/primary hypha septum. Secondary hyphae are narrower than primary hyphae, form their proximal septum some distance along the hypha, develop asynchronously, and proliferate to form the intercellular mycelium. Infection processes and epidermal stripping are discussed.     

Ultrastructure of the Penetration and Infection of Pansy Roots by Thielaviopsis basicola

ABSTRACT Transmission electron microscopy was used to study the penetration and infection of pansy roots by Thielaviopsis basicola. Events observed in 7- to 10-day-old roots produced on moist filter paper differed slightly from those in roots from 4-week-old plants washed free of potting media prior to inoculation. By 3 h postinoculation (PI), epidermal cells of roots produced on filter paper exhibited aggregated cytoplasm and papilla formation in response to germ tube tips. The presence of callose in papillae was demonstrated using immunogold labeling. Papilla formation was not effective in preventing host cell penetration. A slender infection hypha emerged from a germ tube tip and grew through a papilla. Its tip then expanded to form a globose infection vesicle. By 6 h PI, infection hyphae emerged from infection vesicles, and invaded host cells showed signs of necrosis. By 8 h PI, infection hyphae had grown into cortical cells in spite of papilla formation in these cells. By 24 h PI, distinctive intracellular hyphae were present in necrotic cortical cells. In washed roots, most epidermal cells failed to respond to invasion. Hyphae simply grew through these cells and contacted cortical cells that exhibited aggregated cytoplasm and papillae formation. Infection structures similar to those produced in epidermal cells from roots grown on filter paper then formed in cortical cells of washed roots. The fact that T. basicola formed infection structures only in cells that responded to invasion suggests that T. basicola has a more complex relationship with its host than would be expected in a nectrotrophic pathogen. We believe that T. basicola is best described as a necrotrophic hemibiotroph.     

禾顶囊壳小麦变种对小麦种子根的侵染过程

 光镜和电镜观察表明,禾顶囊壳小麦变种(Gaeumannomyces graminis var.tritici,小麦全蚀病菌)对小麦种子根的侵染过程可分为侵入前、侵入表皮层、进入皮层和进入中柱等4个连续阶段。麦根接菌后在15℃下培养,48 h后侵入表皮层细胞,60 h后进入皮层,120 h后进入中柱。病原菌主要以侵染菌丝直接侵入表皮层,表皮细胞间隙和根毛基细胞是主要侵入部位,少数由附着枝侵入。菌丝穿透细胞壁有明显的酶解作用特征,菌丝先端前方胞壁上还产生电子密物质。皮层细胞是病原菌定殖和发展的主要场所,病原菌还能离解胞间层,形成胞外空间,特别有利于菌丝和菌丝束的扩展。在侵入位点的寄主细胞壁和质膜之间,形成多种形状的木质管,其数量与侵入菌丝的数目相对应,但木质管不能阻止菌丝进入细胞。菌丝进入中柱后,可阻塞导管和筛管。小麦细胞发生退行性病变,尤以细胞壁膨大崩坏和早期质壁分离最明显,细胞间隙还产生性质不明的黄色物质。     

Histopathology of the initial stages of the interaction between rose flowers and Botrytis cinerea

The early stages of the interaction between flowers of the cut rose cv. Sonia andBotrytis cinerea was investigated by scanning electron microscopy and light microscopy. Infection of petals by conidial germ tubes evoked a susceptible reaction. In contrast to general findings nutrient addition to the inoculum was not a prerequisite for this phenomenon. At the lower side of germ tube tips the cuticle was penetrated by infection pegs. Already at this early stage of the infection process, the infection sites were macroscopically visible as scattered white spots. After penetration, pegs enlarged to form infection hyphae, which invaded the periclinal wall of outer epidermal cells. At those sites, the petals formed outgrowths of variable appearance at their abaxial side. Thee outgrowths consisted of remanants of collapsed epidermal cells and of infection hyphae. Subsequent intra- and intercellular growth of hyphae led to a collapse of epidermal and mesophyll cells. The symptoms described generally developed within 24 h. After subsequent incubation the lesions became necrotic. Eventually, the necrosis would spread leading to the death of whole petals.     

Infection of resistant and susceptible cultivars of wheat by Pyrenophora tritici-repentis

Conidial germination, appressorial formation. penetration of epidermal walls, formation of intracellular vesicles and growth of intracellular hyphae in epidermal cells occurred within 12 h of inoculation. Hyphae then grew slowly between mesophyll cells for the next 12 h. Some papillae formed beneath appressoria and most infected epidermal cells retained stain by 24 h after inoculation, indicating major changes in cellular physiology. Slight differences between cultivars in some of these events were not related to resistance.
On the second day. intercellular hyphae emerged more extensively from the infection sites into the mesophyll of the susceptible cultivar Banks, and formed significantly larger mycelia than in the resistant cultivar BH1146 by 3-5 days from inoculation. Rapid intercellular growth then continued in the susceptible cultivar but not in the resistant cultivar. Necrotic lesions expanded faster in the susceptible cultivar from day 3. By day 10. most lesions in this cultivar were large and light brown with a conspicuous chlorotic margin but those in the resistant cultivar were small and dark brown with inconspicuous chlorosis.     

Trichomes: interaction sites of tomato leaves with biotrophic powdery mildew pathogens

The present study aimed to explore the possibility of using the type I trichomes of tomato (Solanum lycopersicum) to monitor the infection processes of powdery mildews by microscopy. Individual trichome cells of two tomato genotypes were inoculated with pathogenic and non-pathogenic powdery mildew species, Pseudoidium neolycopersici, Erysiphe trifoliorum and Podosphaera xanthii. On the trichome cells of the tomato cultivar Moneymaker, hyphae of the pathogenic Pseudoidium neolycopersici (isolates KTP-03 and KTP-04) grew vigorously; whereas hyphal growth of the non-pathogenic Erysiphe trifoliorum and Podosphaera xanthii ceased after appressorium formation, which was associated with papilla formation and hypersensitive cell death, respectively. Similar infection processes of the tested powdery mildews were seen in Moneymaker epidermal cells. Therefore, tomato trichomes are suitable for analysing, at individual cell level, the infection processes of different pathotypes of powdery mildews and for observing the cytological responses of plants by non-pathogenic powdery mildews. On the other hand, it was observed that both isolates KTP-03 and KTP-04 failed to produce conidiophores on the hyphae elongating on Moneymaker trichomes. Similarly, no conidiophores were produced on the hyphae elongating on trichomes of Solanum peruvianum LA2172, which is resistant to KTP-03 and susceptible to KTP-04. Interestingly, delayed cell death occurred in LA2172 epidermal cells, which were attacked by KTP-03 hyphae elongating from trichomes and conidiophores were formed on new hyphae growing from the leaf epidermal cells. Thus, leaf trichomes and epidermal cells of the wild tomato species LA2172 reacted differently to the avirulent isolate KTP-03.     

Effect of carpropamid on secondary infection by rice blast fungus

Carpropamid (WIN™, KTU 3616) provides good control of leaf and panicle blast by ‘one-shot’ nursery-box treatment. It inhibits melanin biosynthesis in appressorial cells of Pyricularia oryzae, making them hyaline. Penetration by infection hyphae from the hyaline appressoria into rice epidermal cells is substantially hindered. In addition, the spread of rice blast spores from primary lesions to the other parts of the plant leading to secondary infection is largely prevented when the plants are treated with carpropamid by spray or water surface application. Secondary infection was simulated in a glass chamber fitted with an ultrasonic humidifier. On treated plants, many blast spores formed in the lesions, but the number of air spora that were dispersed from the lesions decreased significantly. A similar suppression of the spore liberation was observed in vitro when lesions on rice leaf segments, or discs from Pyricularia cultures on oatmeal agar were treated with the chemical. Spores from treated lesions or from the cultures on oatmeal agar amended with the chemical germinated normally and produced well-melanized appressoria on cellophane membranes. In addition, the spores proved to be fully pathogenic towards rice seedlings, producing normal disease symptoms. These results strongly suggest that carpropamid reduces the secondary infection of rice by Pyricularia by specifically hindering spore liberation. © 1999 Society of Chemical Industry     

Histopathology of Infection and Colonization of Susceptible and Resistant Port-Orford-Cedar by Phytophthora lateralis

ABSTRACT Port-Orford-cedar (POC) root disease, caused by Phytophthora lateralis, continues to kill POC in landscape plantings and natural forests in western North America. POC trees resistant to P. lateralis have been identified and propagated. Cytological observations of P. lateralis in susceptible and resistant roots and stems were made with light and transmission electron microscopy to identify resistance mechanisms. No differences in infection pathway and initial colonization were observed between susceptible and resistant roots, although there were differences in the rate and extent of development. Germ tubes formed appressoria, and penetration hyphae grew either between or directly through epidermal cell walls; inter- and intracellular hyphae colonized the root cortex. In susceptible roots, hyphae penetrated into the vascular system within 48 h of inoculation. In contrast, hyphae in roots of resistant seedlings grew more slowly in cortical cells and were not observed to penetrate to the vascular tissues. In resistant roots, infection was marked by general thickening of cortical cell walls, wall appositions around penetrating hyphae, collapse of cortical cells, and accumulation of osmophillic granules around hyphae. In susceptible stems, hyphae grew inter- and intracellularly in all cells of the secondary phloem except fiber cells, but were concentrated in sieve and parenchyma cells in the functional phloem. The pattern of penetration and colonization of hyphae was similar in the resistant stems, except that hyphae were found in the fiber cells of the xylem. In resistant stems, there were fewer hyphae in the functional phloem, and cytological changes such as damaged nuclei and disintegrated cytoplasm were evident. Structural changes in resistant stems included collapsed cells, wall thickening, secretory bodies, apposition of electron dense materials, and crystals in cell walls.     

梨黑星病菌寄生部位及致病作用研究

 组织透明及扫描电镜研究结果表明:梨黑星病菌在叶片上主要寄生于表皮细胞和叶脉薄壁组织细胞的表面,沿细胞表面扩展。病原菌并非只在角质层与表皮细胞间生长,而且在表皮细胞和叶脉薄壁组织细胞的细胞间寄生。在大量的观察中,没有发现侵入到细胞内菌丝或菌丝变态结构。梨黑星病菌不寄生叶肉细胞,但能导致叶肉细胞病变。由于黑星病菌的寄生性既不同于专性寄生,也不同于杀生寄生,才使梨黑星病的潜育期和产孢期都长于这两类真菌病害。这是认识和研究梨黑星病发病特点和流行规律的基础。     

Histological and ultrastructural characterization of the leaf infection events of Colletotrichum fructicola on Malus domestica ‘Gala’

Glomerella leaf spot (GLS), characterized by black necrotic spots and severe defoliation, is a destructive foliar disease of apple. Widely grown cultivars such as Gala and Golden Delicious are highly susceptible to GLS. Currently, the infection biology of the causal pathogen, Colletotrichum fructicola, on apple leaves is unclear. In the present study, the penetration and colonization processes of C. fructicola were characterized on apple (cv. Gala) leaves using light and transmission electron microscopy. C. fructicola conidia produced germ tubes 4 hours post-inoculation (hpi) and appressoria at 8 hpi. In melanized appressoria, funnel-shaped appressorial cones formed around the penetration pore. At 12 hpi, C. fructicola produced secondary conidia. After penetration, C. fructicola began to develop infection vesicles at 36 hpi. At 48 hpi, the primary hyphae of C. fructicola were produced from infection vesicles within host epidermal cells; the host epidermal cell plasma membrane remained intact, indicating a biotrophic association. Subsequently, secondary hyphae penetrated epidermal cells and destroyed cell components, initiating necrotrophic colonization. C. fructicola also produced biotrophic subcuticular infection vesicles and hyphae. Together, these results demonstrate that C. fructicola forms special infection structures and colonizes apple leaves in a hemibiotrophic manner, involving intracellular as well as subcuticular colonization strategies. Detailed characterization of the infection process of C. fructicola on apple leaves will assist in the development of disease management strategies and provide a foundation for studies of the molecular mechanism of the C. fructicola–apple leaf interaction.