Fungal and plant gene expression during synchronized infection of tomato leaves by Botrytis cinerea

An inoculation procedure was developed to obtain efficient and synchronous infection on detached tomato leaves by Botrytis cinerea. In spray-inoculated leaves incubated at 20 °C, the infection process consisted of three phases: the formation of primary necrotic lesions (until 20 hpi), a quiescent phase (20-72 hpi), and the expansion of a proportion of the primary lesions (from 72 hpi onwards), resulting in full tissue maceration. At 4 °C, the infection progressed slowly but steadily without inducing necrotic responses in the host. The actin and -tubulin genes of B. cinerea were cloned, characterized and used as probes on blots containing RNAs from leaves at various stages of the infection. The genes displayed a similar expression pattern throughout the infection and the hybridization signal reflected the amount of fungal biomass. The actin mRNA accumulated to higher levels than the -tubulin mRNA. Tomato PR protein mRNAs (chitinase, -1,3-glucanase and PR-1) were induced during the infection, albeit with different kinetics and to different levels. At 20 °C, -1,3-glucanase and PR-1 mRNAs were induced more rapidly than chitinase mRNAs. At 4 °C, mRNAs encoding extracellular -1,3-glucanase and intracellular, as well as extracellular chitinase were hardly induced.     

Histological characterization of the early-stage infection events of Setosphaeria turcica in maize

Northern corn leaf blight (NCLB) caused by Setosphaeria turcica is a major foliar disease of maize. The early-stage infection events of this pathogen on maize leaves are unclear. We investigated the optimum temperature for conidial germination and appressorium formation, and characterized penetration and growth of S. turcica in maize leaf sheath and onion epidermis cells, including use of histological staining to assess plant cell viability. The results showed that the optimum temperature for conidial germination and appressorium formation was 20°C. On the maize leaf sheath, the appressoria were formed by germinated conidia, and penetration on the epidermal cells occurred at 8 h postinoculation (hpi). Round vesicles developed beneath the appressoria. Between 16 and 24 hpi, the branched invasive hyphae invaded three to five adjacent cells at most infection sites. The invasive hyphae tended to move along the cell wall and crossed from one cell to another. In the onion epidermis cells, the appressoria formed at 8 hpi, and in most cases the epidermal cells were penetrated through the juncture of the cell walls. At 16–24 hpi, the primary hyphal terminus swelled to a vesicle. The maize leaf sheath cells died at 8 hpi, whereas the onion cells did not. Our findings documented in detail the penetration and invasive hyphal growth in maize leaf sheath and onion epidermis, as well as viability of plant cells, at the early stages of infection, and provide a foundation for elucidating the underlying mechanism of S. turcica–maize interactions.     

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.     

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.     

Infection process of <Emphasis Type="Italic">Pseudocercospora musae</Emphasis> on banana leaf

Yellow Sigatoka that is caused by Pseudocercospora musae is an important banana disease. The aim of this study was to elucidate the infection process of P. musae in banana leaves by scanning electron microscopy. Leaf samples were inoculated on the abaxial surface with P. musae and then analysed at 12, 24, 36, 48, 72, 96, 120, 144, and 168 h post inoculation (hpi) and at 36 and 50 days post inoculation (dpi). The conidia were found to be germinated between 24 and 36 hpi and penetrated through the stomata between 96 and 120 hpi, or more generally from 144 hpi. P. musae colonized the spongy parenchyma at 36 dpi and the palisade parenchyma at 50 dpi. Sporulation occurred at 50 dpi on the adaxial surface of leaves through the emergence of conidia on conidiophores through the stomata. Considering the importance of yellow Sigatoka in banana production, our results provide a better understanding of the life cycle of the fungus for treatment processes.     

Infection processes of Ustilaginoidea virens during artificial inoculation of rice panicles

Ustilaginoidea virens is a ubiquitous plant pathogen that causes rice false smut disease, one of the most destructive diseases of rice (Oryza sativa L.) production. However, data concerning the effect of inoculation on disease development and the infection process of this pathogen are not comprehensive. In this study, the developmental processes of U. virens in rice panicles were characterized using an enhanced green fluorescent protein (EGFP) labelled strain. A mixture of hyphae and conidia of U. virens was used to inoculate rice panicles by leaf sheath injection during the booting stage of rice plants grown in a greenhouse. The panicles were assessed to determine the relationship between artificial inoculation and disease occurrence. Increasing volumes of inocula (0.2, 0.5, 1, and 2 ml of a mixture of hyphae-fragment and 2?×?10⁶ conidia/ml suspension) caused more severe infections, and small differences were also observed for the different inoculation sites at the base, apex and mid-point of rice panicles. The optimum inoculation condition was 1–2 ml inoculum injected into the mid-point of rice panicles. Spikelet samples were collected as the disease progressed and observed by confocal laser scanning microscopy and scanning electron microscopy. The images collected showed that the primary site of U. virens colonization was at the base of the filaments with the inner spikelets becoming infected by hyphae at 24 h post inoculation (hpi). The accumulation of hyphae reached its highest level at 168 hpi, before the rice heading stage, as the infection extended upward from basal filaments to the anther apex, and then enclosed all the floral organs to produce a velvety smut ball.     

Infection process of <Emphasis Type="Italic">Gilbertella persicaria</Emphasis> in papaya (<Emphasis Type="Italic">Carica papaya</Emphasis> L.) fruits

Gilbertella persicaria is a pathogenic fungus recently reported as a causative agent of soft rot in papaya fruits. Here the interactions between G. persicaria and papaya fruits was analyzed under laboratory conditions using histological techniques and optical microscopy to elucidate the process of pathogenesis. Healthy and disinfested fruits of papaya cv. Maradol were also inoculated with a suspension of sporangiospores of G. persicaria. Tissue sections were cut, which were subjected to differential staining with safranin-fast green for different times. Sporangiospores presumably adhered to the cuticle of the fruit by 3 h post inoculation (hpi) and germinated by 6 hpi; invasive intracellular hyphae were growing in host cells by 9 hpi. By 15 hpi, fruit epidermis was macerated, presumably by enzymatic activity reported for mucoral fungal species and appeared as a wet-looking lesion on the cuticle. Fruit mesocarp was colonized by 30 hpi, and asexual reproduction structures had formed by 48 hpi. This process of infection and disease development of G. persicaria in papaya fruits corresponds to that used by pathogens with a necrotrophic lifestyle.     

内质网应激因子NAC089突变体的创建及其对病毒侵染胁迫的响应

 有研究表明烟草花叶病毒(Tobacco mosaic virus, TMV)或黄瓜花叶病毒(Cucumber mosaic virus, CMV)侵染烟草能够激活转录因子NbNAC089,从ER膜移至细胞核。为进一步阐释内质网应激因子NbNAC089对病毒侵染胁迫的响应机制,利用CRISPR/Cas9技术构建敲除载体,经烟草遗传转染获得NbNAC089基因突变植株。植株接种病毒后采用qRT-PCR检测病毒CP基因和寄主UPR基因的表达。结果表明:CRISPR/Cas9系统定点敲除NbNAC089基因后,目的基因靶位点序列有碱基的置换与缺失。正常生长条件下,转基因植株与野生型无差异。植株接种TMV-GFP后24~96 h,突变体中UPR基因(BiP、bZIP28、bZIP60)的表达量显著高于野生型;接种TMV-GFP后2~6 d突变体中病毒的积累量和扩展速度显著高于野生型。表明NbNAC089为UPR的抑制因子,对病毒增殖具有负调控作用。     

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.     

Light and Scanning Electron Microscopy Studies on the Infection of Oriental Lily Leaves By Botrytis Elliptica

Light, scanning electron and fluorescent microscopy were used to observe the infection process of Botrytis elliptica on leaves of oriental lily (cv. Star Gazer). At 20 °C and 100% relative humidity, conidia germinated on both adaxial and abaxial foliar surfaces, but germ tubes failed to invade epidermal cells on the adaxial surface. On abaxial surfaces, short (< 20 m) swollen germ tube appressoria penetrated through stomatal openings (19%), through the epidermis near guard cells (52%), or directly through epidermal cells (29%). Esterase activity was detected on germ tubes and conidia after 6 h of incubation, and deformation of the cuticle on abaxial surfaces of lily was observed surrounding infection sites. By 3 h after inoculation, almost 70% of the conidia had germinated, but no penetration was observed. At 6 h after inoculation, almost one-third of germinated conidia had penetrated epidermal cells, and water-soaked lesions were associated with 20% of the penetrations. By 9 h after inoculation, approximately 60% of the germinated conidia had penetrated plant tissues, and water-soaked lesions were associated with 60% of the infections. Fluorescent microscopy with a specific fungal stain allowed assessment of successful infection and visualization of sub-epidermal hyphae. We conclude that penetration of abaxial foliar surfaces of oriental lilies by B. elliptica occurs via short swollen germ tube appressoria mostly near stomata.     

水稻纹枯病发病过程PR1和PBZ1的表达动态

 采用从福建省稻田分离纯化的纹枯病菌(Rhizoctonia solani)菌株FJ-15接种籼稻9311,分析了水稻在纹枯病菌侵染致病过程中,编码病程相关蛋白的基因表达动态,并观察了症状的变化。Northern blot分析表明:PR1在接种12 h后开始表达,在之后的4个时间段其表达量逐步增强;而PBZ1也在12 h开始表达,在48 h表达量激剧增强几乎与72 h表达量相当。组织和症状观察表明,接种12 h后叶鞘表面菌丝纵横分枝,接种36 h后出现零星病斑,接种48 h后表现典型的受害症状,接种72 h后病斑继续扩大,并可蔓延到非接种叶鞘。结果表明,PR1和PBZ1的表达与水稻和纹枯病菌亲和互作的过程存在对应关系。     

Systemic induction of chitinase activity and resistance in melon plants upon fungal infection or elicitor treatment

Melon plants locally infected with Colletotrichum lagenarium display a marked increase in chitinase activities (exo- and endo-activities) throughout the whole plant. This increase begins 3 days after inoculation in the inoculated cotyledon, and then occurs sequentially in the non-infected tissues.Both fungal elicitors and plant endogenous elicitors induce a rapid increase in chitinase activity in the treated cotyledon. In other organs, chitinase activity is stimulated, to a lesser extent and after a lag period, only by fungal elicitors.The earlier, more rapid, systemic induction of chitinase activity, produced by treatment with the fungal elicitor is correlated by the increased resistance of the tissues to infection by the pathogen.     

Movement of <Emphasis Type="Italic">Cucumber Mosaic Virus</Emphasis> Is Restricted at the Interface between Mesophyll and Phloem Pathway in <Emphasis Type="Italic">Cucumis figarei</Emphasis>

Viral movement in the leaf tissues of a resistant host, Cucumis figarei, inoculated with the pepo strain of Cucumber mosaic virus (CMV) and incubated at 24°C or 36°C was investigated by fluorescence in situ hybridization (FISH), leaf-press blotting, tissue printing and immunogold-silver staining techniques. Observation by FISH revealed that at 24°C most infection sites with CMV at 0.01 mg/ml or 0.1 mg/ml were limited to a single cell during the incubation period, that the number of infection sites increased from 24hpi (hours post inoculation) to 80 hpi in the leaves inoculated with CMV at 0.5 mg/ml, and that the size as well as the number of infection sites rapidly increased with time in the leaves inoculated with CMV at 2.0 mg/ml. These results suggested that one factor for the resistance of C. figarei at 24°C might be an inhibition of viral movement in and out of the infection sites. Leaf-press blotting and tissue blotting indicated that CMV remained in the infection sites at 24°C, whereas it spread from the inoculated leaves to other parts of the plants through vascular systems at 36°C. Immunogold-silver staining demonstrated that at 24°C CMV infected bundle sheath (BS) cells in minor veins, whereas at 36°C it invaded not only BS cells, but also phloem parenchyma (PP)/ companion cell (CC) or PP/intermediary cell (IC) complexes in minor veins in the regions with chlorotic symptoms. These results indicated that at 24°C CMV had difficulty in passing through the interface between BS and PP/CC or PP/ IC complexes and that viral entry from mesophyll to the phloem pathway was inhibited in the inoculated leaves. Received 26 August 1999/ Accepted in revised form 14 December 1999     

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.     

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.     

Infection with Rhizoctonia solani induces defense genes and systemic resistance in potato sprouts grown without light

Rhizoctonia solani is an important soilborne and seedborne fungal pathogen of potato (Solanum tuberosum). The initial infection of sprouts prior to emergence causes lesions and may be lethal to the sprout or sprout tip, which results in initiation and compensatory growth of new sprouts. They emerge successfully and do not suffer significant damage. The mechanism behind this recovery phenomenon is not known. It was hypothesized that infection may induce pathogen defense in sprouts, which was investigated in the present study. Tubers were sprouted in cool and moist conditions in darkness to mimic conditions beneath soil. The basal portion of the sprout was isolated from the apical portion with a soft plastic collar and inoculated with highly virulent R. solani. Induction of defense-related responses was monitored in the apical portion using microarray and quantitative polymerase chain reaction techniques at 48 and 120 h postinoculation (hpi) and by challenge-inoculation with R. solani in two experiments. Differential expression of 122 and 779 genes, including many well-characterized defense-related genes, was detected at 48 and 120 hpi, respectively. The apical portion of the sprout also expressed resistance which inhibited secondary infection of the sprouts. The observed systemic induction of resistance in sprouts upon infection with virulent R. solani provides novel information about pathogen defense in potato before the plant emerges and becomes photosynthetically active. These results advance our understanding of the little studied subject of pathogen defense in subterranean parts of plants.     

Verticillium dahliae Modifies the Concentrations of Proline, Soluble Sugars, Starch, Soluble Protein and Abscisic Acid in Pepper Plants

The objective of this research was to study the levels of some organic solutes, such as total protein, total soluble sugars, starch and proline in leaves, as well as abscisic acid concentration in xylem of pepper plants inoculated with Verticillium dahliae Kleb. Healthy and inoculated plants were always kept well watered. Measurements were made at time intervals after inoculation. Leaf water potential rapidly decreased as a consequence of fungal infection. However, relative water content in leaves only changed significantly from day 20 after inoculation, and such decreases coincided with a sharp build up of proline and total soluble sugars in leaves. Starch and protein levels, as well as abscisic acid concentration in xylem, declined in healthy and inoculated peppers as they became older. However, such decreases were more pronounced in infected plants, especially soon after inoculation. Results suggest that proline and total soluble sugars accumulation could be sensors of the damage caused by the fungal infection.     

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。