Histopathological assessment of wheat seedling tissues infected by Fusarium pseudograminearum

Histopathological assessment of infection by the crown rot pathogen Fusarium pseudograminearum in wheat seedling tissues was performed using fluorescence microscopy. The coleoptiles and leaf sheaths of four host cultivars of differing susceptibility were examined. Leaf sheaths were most frequently penetrated via stomata, indicated by initial lesions forming at the guard cells. Internally, cell wall penetration was facilitated by penetration structures which appeared as hyphal swellings or septate foot‐shaped appressoria. Colonization of leaf sheaths resulted in the re‐emergence of hyphae from stomata on both surfaces of the sheath. These hyphae are hypothesized to have two major roles; first as exploratory hyphae for colonization of new tissues, and secondly as sites of profuse conidial production. The formation of conidia on the leaf sheath surface was only recorded on the most susceptible bread wheat genotype. No other major differences in host–pathogen interactions were observed among these cultivars. Almost all cell types in the leaf sheath tissues were extensively colonized, except for the vascular bundles and silica cells. This investigation provides the first comprehensive assessment of F. pseudograminearum infection structures and growth patterns during the infection of wheat seedlings.     

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.     

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.     

Effects of panicle development stage and temperature on rice panicle blast infection by Magnaporthe oryzae and visualization of its infection process

Panicle blast, caused by the fungus Magnaporthe oryzae (syn. Pyricularia oryzae), directly contributes to yield loss in the field. The effects of panicle development stage and temperature on panicle blast were studied and the infection process of M. oryzae in panicles was visualized. Rice panicles at different development stages from three rice cultivars were inoculated with a conidial suspension in vitro. The rice cultivar Lijiangxintuanheigu was highly susceptible to panicle blast at 5 days postinoculation (dpi) when the pulvinus distance was 15–20 cm. Nanjing 9108 was moderately susceptible to panicle blast when the pulvinus distance was 8–10 cm, but Yliangyou 800 was resistant. The effect of temperature on panicle blast was determined under 22–35 °C temperature treatments. Inoculated panicles placed at temperatures of 28 and 30 °C showed the highest lesion grade based on lesion length at 5 dpi. The infection process of M. oryzae in rice panicles was observed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). M. oryzae initially formed the appressorium to invade through the epidermis of rice panicles at 24 hours postinoculation (hpi). As the disease progressed, the invasive hyphae formed dense mycelial networks in the inner parenchyma cells at 60 hpi. Our results will contribute to the understanding of panicle development stage and temperature effects on panicle blast and improve resistance evaluation methods. Additionally, visualization of the infection process by CLSM and TEM are valuable methods to observe M. oryzae invasive hyphae inside rice panicle cells.     

荸荠茎点霉秆枯病菌侵染过程的超微观察

<正>荸荠(Eleocharis dulcis),又称马蹄,为莎草科多年生草本植物,是一种具有食用和药用价值的水生蔬菜。近年来,随着荸荠在我国种植面积的不断扩大,病害发生也呈逐年上升趋势。荸荠茎点霉秆枯病是2009年在湖北省荸荠产区发现的一种新病害,由Phoma bellidis侵染引起,该病在湖北省团风地区发生尤为严重,对荸荠的产量和品质造成严重影响;病害一般在8~12月发生,发病初期在荸荠茎秆上产生圆形或梭形红褐色小斑,随后病斑沿茎     

立枯丝核菌侵染玉米的研究

 用获自水稻及玉米的立枯丝核菌(Rhizoctonia solani Khn) AG-11A接种玉米发生典型纹枯症状,其致病力显著强于AG-4。玉米拔节期,上位叶鞘抗性较强,抽雄及抽丝期抗性减弱,下位叶鞘无论在拔节期或抽雄、抽丝期,均较上位叶鞘感病。接种玉米后8小时,形成侵染垫及附着胞,从这些结构上形成侵入钉侵入,AG-4侵染上位叶鞘时,常以菌丝直接穿透表皮或从气孔侵入。在去掉菌体的叶鞘表面,发现有周边光滑或稍破损的侵入孔。接种后12小时,在叶鞘细胞中发现菌丝,它们在穿过细胞壁进入邻近细胞时,明显变细。接种后16小时,新生出的菌丝从气孔成丛出现。     

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.     

A histological assessment of the infection strategy of Exserohilum turcicum in maize

Northern leaf blight is a lethal foliar disease of maize caused by the fungus Exserohilum turcicum. The aim of this study was to elucidate the infection strategy of the fungus in maize leaves using modern microscopy techniques and to understand better the hemibiotrophic lifestyle of E. turcicum. Leaf samples were collected from inoculated B73 maize plants at 1, 4, 9, 11, 14 and 18 days post-inoculation (dpi). Samples were prepared according to standard microscopy procedures and analysed using light microscopy as well as scanning (SEM) and transmission electron microscopy (TEM). Microscopic observations were preceded by macroscopic observations for each time point. The fungus penetrated the leaf epidermal cells at 1 dpi and the disease was characterized by chlorotic leaf flecks. At 4 dpi the chlorotic flecks enlarged to form spots, and at 9 dpi hyphae were seen in the epidermal cells surrounding the infection site. At 11 dpi lesions started to form on the leaves and SEM revealed the presence of hyphae in the vascular bundles. At 14 dpi the xylem was almost completely blocked by hyphal growth. Hyphae spread into the adjacent bundle sheath cells causing cellular damage, characterized by plasmolysis, at 18 dpi and conidiophores formed through the stomata. Morphologically, lesions started to enlarge and coalesce leading to wilting of leaves. This study provides an updated, detailed view of the infection strategy of E. turcicum in maize and supports previous findings that E. turcicum follows a hemibiotrophic lifestyle.     

疏水表面诱导橡胶树炭疽菌侵染结构的发育分化过程

为了解橡胶树2种炭疽病菌的侵染结构发育分化过程,采用平板菌落生长速率法测定了3株胶孢炭疽菌Colletotrichum gloeosporioides和3株尖孢炭疽菌C.acutatum的菌丝生长速率,测量其分生孢子大小,显微观察2种炭疽菌在疏水表面诱导下侵染结构的发育分化过程。结果表明,胶孢炭疽菌菌丝生长速率为0.96~1.36 cm/d,显著高于尖孢炭疽菌的菌丝生长速率0.72~0.89 cm/d,但二者分生孢子大小无显著差异。在疏水表面诱导下,2种炭疽菌分生孢子在接种2~6 h后开始萌发,12 h孢子萌发率为71.70%~88.05%,13~16 h开始分化附着胞,24 h附着胞形成率为48.99%~70.74%,36 h菌丝诱发形成大量附着枝,48 h后分生孢子产生的次生菌丝也可诱发形成附着枝,附着枝呈圆形、姜瓣形、梨形或不规则形。分生孢子极易产生,可在菌丝顶端成簇或菌丝侧面排列产生,也可由分生孢子形成的芽管产生,或在芽管分化附着胞过程分枝形成分生孢子;附着胞多着生于芽管顶端,少数附着胞顶端可继续萌发类似短芽管结构,再次分化形成可黑色化的次级附着胞。表明橡胶树2种炭疽菌不同菌株间分生孢子萌发时间、孢子萌发率、附着胞形成时间和形成率有一定差异,但种间无明显差异;橡胶树炭疽菌分生孢子极易形成,在疏水表面容易分化形成附着胞和附着枝,说明具有极强的适生性。     

Transgenic rice plants that over-express the mannose-binding rice lectin have enhanced resistance to rice blast

Mannose-binding rice lectin (MRL), which is almost identical to the salt-induced protein SalT, binds to mannose and glucose residues. Expression of the MRL gene in response to infection with Magnaporthe oryzae, the rice blast fungus, was stronger in the incompatible interaction than in the compatible. Transgenic rice plants that constitutively over-expressed MRL strongly suppressed the growth of invasive hyphae of the fungus on leaf sheaths and the development of typical susceptible-type lesions on leaf blades, but did not affect penetration by the fungus in comparison with the wild-type. On a polycarbonate plate, purified recombinant MRL inhibited conidial attachment and appressorium formation but not conidial germination. These results suggest that MRL may play an essential role in disease resistance by suppressing development of M. oryzae in situ.     

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。     

Histology of waxflower (Chamelaucium spp.) flower infection by Botrytis cinerea

Botrytis cinerea infects waxflower (Chamelaucium spp.) flowers and can induce them to abscise from their petioles before disease becomes evident. Botrytis cinerea infection of flowers was studied on two waxflower cultivars by light and electron microscopy. Pot‐grown waxflower flowers were harvested, inoculated with aqueous suspensions of B. cinerea conidia, incubated at 20–22°C and >95% RH and examined within 96 h post‐inoculation (hpi). Conidial germination on petals started 4 hpi, penetration via germ tube tips was 6 hpi and protoappressoria were formed 8 hpi. Germination on petals approximately doubled every 4–6 h to 18 hpi. Conidial germination was ca. 50% at 22–24 hpi. Botrytis cinerea infected most waxflower flower organs, including petals, anthers and filaments, stigma and hypanthium, within 24 hpi. Hyaline and lobate appressoria were observed 36 hpi. Infection cushions on stamen bases were formed 36 hpi by saprophytic hyphae that originated from anthers. This infection process can give rise to tan‐coloured symptoms typical of botrytis disease that radiate from this part of the flower. Subcuticular hyphae were present at high density near stamen bases and evidently resulted from multiple penetrations from single infection cushions. The subcuticular hyphae grew within the hypanthium and towards the centre of the floral tube. When flower abscission occurred, floral tube tissues close to the abscission zone remained uninfected. This observation infers possible transmission of a signal (e.g. ethylene) upon B. cinerea infection. Thus, B. cinerea causes flower abscission apparently as a defence response.     

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.     

Infection of onion leaves by Alternaria porri and Stemphylium vesicarium and disease development in controlled environments

Infection of onion by Alternaria porri and Stemphylium vesicarium was investigated under a range of controlled temperatures (4–25°C) and leaf wetness periods (0–24 h). Conidia of A. porri and S. vesicarium germinated within 2 h when incubated at 4°C. Terminal and intercalary appressoria were produced at similar frequencies at or above 10°C. The maximum number of appressoria was produced after 24 h at 25°C. Penetration of leaves by both pathogens was via the epidermis and stomata, but the frequency of stomatal penetration exceeded that of epidermal penetration. There was a strong correlation ( R ² > 90%) between appressorium formation and total penetrations at all temperatures. Infection of onion leaves occurred after 16 h of leaf wetness at 15°C and 8 h of leaf wetness at 10–25°C, and infection increased with increasing leaf wetness duration to 24 h at all temperatures. Interruption of a single or double leaf wetness period by a dry period of 4–24 h had little effect on lesion numbers. Conidia of A. porri and S. vesicarium separately or in mixtures caused similar numbers of lesions. Alternaria porri and S. vesicarium are both potentially important pathogens in winter-grown Allium crops and purple leaf blotch symptoms were considered to be a complex caused by both pathogens.     

Cytology of infection of apple leaves by Diplocarpon mali

Diplocarpon mali, the causal agent of Marssonina leaf blotch of apple, causes severe defoliation during the growing season. Little information is available on the mode of infection and the infection process. In this study, the infection strategies of D. mali in apple leaves were investigated using fluorescence and electron microscopy. Conidia attached to leaf surface apparently by mucilage and germinated on both sides of leaves 6 h post-inoculation (hpi). The pathogen penetrated the cuticle by infection pegs formed either in germ tubes or appressoria in 6 hpi, and then formed haustoria in host epidermal and mesophyll cells accompanied by extension of subcuticular and intercellular hyphae. Five days post-inoculation (dpi), the intracellular hyphae were observed. At the same time, the subcuticular hyphal strands (SHS) were produced as a means for fast expansion and reproduction. About 7 dpi, acervuli formed on inoculated leaves. This was the first observation that D. mali formed haustoria and SHS as infection strategies. Our results suggest that D. mali may behave like a hemibiotroph, which can use both biotrophic and necrotrophic strategies to establish infections on apple leaves.     

Expression of resistance to Mycosphaerella pinodes in Pisum sativum

The infection of three resistant and two susceptible inbred lines of Pisum sativum by Mycosphaerella pinodes is described for the first time. Two types of resistance, one expressed in epicotyls and one in leaves, were found in all three resistant lines. On epicotyls of susceptible lines, abundant appressoria and penetrations occurred after a short period of hyphal growth. On epicotyls of resistant lines, hyphae grew extensively but rarely formed appressoria, and these failed to penetrate the cuticle. Attempted penetration was associated with the rapid death of 2–6 epicotyl cells, resembling a hypersensitive reaction. In contrast, resistance of leaves, which was only expressed after penetration, involved localization of the fungus by a mechanism involving delayed leaf cell death. It is suggested that a combination of these two types of resistance might provide effective protection against M. pinodes.     

Novel infection strategies of Colletotrichum acutatum on ripe blueberry fruit

The infection and colonization process of Colletotrichum acutatum on ripe blueberry fruit from two cultivars with different susceptibility to anthracnose were examined using light and confocal laser scanning microscopy. Ripe fruit from susceptible cv. Jersey and resistant cv. Elliott were drop-inoculated with a conidial suspension of C. acutatum, and epidermal peels were evaluated at selected times after inoculation and incubation. Results from pre-penetration studies demonstrated that there were significant differences in the rate of formation of melanized appressoria between the two cultivars, with the rate of formation being faster in the susceptible one. In both cultivars, penetration by the pathogen occurred via appressoria 48 h post-inoculation (hpi). However, in the susceptible cv. Jersey, C. acutatum then adopted an intracellular hemibiotrophic-like infection strategy, whereas in the resistant cv. Elliott subcuticular intramural-like infection occurred. In cv. Jersey by 108 hpi, intracellular growth of the pathogen led to the formation of numerous acervuli, with orange conidial masses. By 120 hpi, the conidial masses had coalesced covering the entire inoculated area. In cv. Elliott, acervuli were not seen until 144 hpi and contained few conidia. These results demonstrate for the first time the ability of C. acutatum to adopt a different infection and colonization strategy depending on the susceptibility of the host tissue being colonized.     

Possible roles and functions of LPL1 gene encoding lysophospholipase during early infection by Magnaporthe grisea

The rice blast fungus Magnaporthe grisea differentiates appressoria, which are required to attack its rice plant host. Clone A26, tentatively named LPL1, was previously found to be homologous to the known lysophospholipase genes from our subtractive cDNA library. The LPL1 protein had a consensus motif (GxSxG) and a catalytic triad (S, D, H) of esterases in the deduced amino acid sequence, and the protein expressed in Escherichia coli had lysophospholipase activity. To clarify the functions and possible roles of LPL1, the gene was disrupted by targeted gene replacement. The ΔLPL1 mutants formed fewer appressoria on the hydrophobic surface of GelBond film, and the appressoria had reduced turgor pressure and penetration into cells of the leaf sheath. The ΔLPL1 mutants and wild-type differentiated normal appressoria on other artificial substrata such as polycarbonate plate and on rice leaf sheath. Cytological analysis of the appressoria indicated that ΔLPL1 mutants had a delay in the disappearance of lipid droplets. These findings imply that LPL1, phospholipid metabolism, or both are involved in glycerol biosynthesis and accumulation to generate turgor pressure in the appressorium. LPL1 was, however, dispensable for full pathogenicity, suggesting that other complementary pathways or similar genes related to phospholipid metabolism probably function in M. grisea.     

Light and scanning electron microscopy studies of the early infection stages of Hymenoscyphus pseudoalbidus on Fraxinus excelsior

The ontogeny and morphology of infection structures associated with the early stages of infection of Hymenoscyphus pseudoalbidus on common ash (Fraxinus excelsior) leaves and leaf petioles were investigated using light and scanning electron microscopy. Ascospores were produced in mature ascocarps and infections on ash leaves were first observed 2 weeks later. Ascospores developed germ tubes, followed by appressorium formation and penetration of epidermal cells on ash leaves and petioles. Chalara fraxinea spores, the anamorph of H. pseudoalbidus, appeared and were arranged in chains, surrounded by a membranous sheath, and varied considerably in size and shape. Host invasion and colonization of all cell types of leaves and petioles were observed using light microscopy. The role of leaves and petioles as sites of infection in the life cycle of H. pseudoalbidus and the disease cycle of ash dieback is discussed.