Germination of Erysiphe gratninis f.sp. hordei conidia on barley leaves

Germination of Erysiphe graminis f.sp. hordei conidia on leaves of several barley cultivars was studied in the laboratory. On both detached leaves and intact plants, within 48 h of inoculation a higher proportion of conidia had germinated on the basal and middle portions of the adaxial leaf surface than on the corresponding portions of the abaxial surface. Such differences between surfaces were not observed near the leaf tip. Similar results were obtained with all the cultivars and growth stages tested, and with five isolates of E. graminis, and are consistent with the observation that there is usually less powdery mildew on the abaxial than the adaxial surface of barley leaves. With most of the barley genotype/mildew isolate combinations tested, within 48 h of inoculation higher proportions of conidia germinated on seedlings and juvenile plants than on older plants. Inherited characteristics which affect spore germination on the leaf surface may be important factors in the development of adult-plant resistance of barley to powdery mildew, particularly in certain genotypes.     

Two sides of a leaf blade: <Emphasis Type="Italic">Blumeria graminis</Emphasis> needs chemical cues in cuticular waxes of <Emphasis Type="Italic">Lolium perenne</Emphasis> for germination and differentiation

Plant surface characteristics were repeatedly shown to play a pivotal role in plant–pathogen interactions. The abaxial leaf surface of perennial ryegrass (Lolium perenne) is extremely glossy and wettable compared to the glaucous and more hydrophobic adaxial surface. Earlier investigations have demonstrated that the abaxial leaf surface was rarely infected by powdery mildew (Blumeria graminis), even when the adaxial surface was densely colonized. This led to the assumption that components of the abaxial epicuticular leaf wax might contribute to the observed impairment of growth and development of B. graminis conidia on abaxial surfaces of L. perenne. To re-assess this hypothesis, we analyzed abundance and chemical composition of L. perenne ab- and adaxial epicuticular wax fractions. While the adaxial epicuticular waxes were dominated by primary alcohols and esters, the abaxial fraction was mainly composed of n-alkanes and aldehydes. However, the major germination and differentiation inducing compound, the C₂₆-aldehyde n-hexacosanal, was not present in the abaxial epicuticular waxes. Spiking of isolated abaxial epicuticular Lolium waxes with synthetically produced n-hexacosanal allowed reconstituting germination and differentiation rates of B. graminis in an in vitro germination assay using wax-coated glass slides. Hence, the absence of the C₂₆-aldehyde from the abaxial surface in combination with a distinctly reduced surface hydrophobicity appears to be primarily responsible for the failure of normal germling development of B. graminis on the abaxial leaf surfaces of L. perenne.     

Infection Process of Cercospora coffeicola on Coffee Leaf

Brown eye spot, caused by Cercospora coffeicola, is an important disease of coffee. Both adaxial and abaxial leaf surfaces were inoculated with a conidial suspension of C. coffeicola. Samples were collected from 4 to 168 h after inoculation and then again at 35 days. Germinated conidia showed positive tropism to stomata where attempted penetrations occurred. Appressoria were not observed. After penetration, C. coffeicola colonized the lacunous parenchyma both inter and intracellularly. Sporulation occurred through or around the stomata. Results from this study provide new insights into the infection process of C. coffeicola on coffee leaf.     

Development of powdery mildew on leaves of several barley varieties at different growth stages

On detached leaves and intact plants of several barley varieties at different growth stages, lower percentages of germinated conidia of Erysiphe graminis f.sp. hordei penetrated the host and initiated infection on the abaxial than adaxial surface. More and larger E. graminis colonies developed on the adaxial surface and these comprised more densely packed hyphae and produced more conidiophores than did colonies on the abaxial surface. These results are consistent with the observation that there is usually more powdery mildew on the adaxial than abaxial surface of barley leaves in the field. Smaller proportions of germinated E. graminis conidia penetrated and infected the host on leaves of adult or near-adult plants than on those of seedlings or juvenile plants. Older plants also supported fewer, smaller and less dense colonies with less sporulation than young plants. The effects of growth stage of the host plant on development of powdery mildew were much greater in some barley varieties, and with some E. graminis isolates, than others.     

Targeted destruction of fungal structures of Erysiphe trifoliorum on flat leaf surfaces of Marchantia polymorpha

In this study, we observed the germination behaviour of airborne conidia from powdery mildews that settle on thalloid surfaces. We inoculated thalli (flat, sheet‐like leaf tissues) and gemmae (small, flat, sheet‐like leaf tissues that propagate asexually via bud‐like structures) of the common liverwort (Marchantia polymorpha) with conidia from tomato powdery mildew (Oidium neolycopersici; KTP‐02) and red clover powdery mildew (Erysiphe trifoliorum; KRCP‐4N) and examined their germination and subsequent appressorium formation under a high‐fidelity digital microscope. Conidial bodies and germ tubes of the inoculated KRCP‐4N conidia were destroyed on both the thalli and gemmae. The destruction of these fungal structures was observed only for KRCP‐4N conidia inoculated onto M. polymorpha on both leaf surfaces. No differences in destruction of the KRCP‐4N fungal structures between thalli and gemmae were observed. At 4 h post‐inoculation, destruction of the germ tube tip was observed when it reached the gemmae leaf surface. At 6 h post‐inoculation, the conidial bodies and germ tubes were destroyed. In contrast, KTP‐02 conidia were not destroyed and formed normal, well‐lobed appressoria on the surface of M. polymorpha gemmae.     

The Infection Process of Pestalotiopsis Longisetula Leaf Spot on Strawberry Leaves

Pestalotia leaf spot, caused by the fungus Pestalotiopsis longisetula Guba, has become the major disease affecting strawberry production in Brazil. Strawberry seedlings with 4–5 leaves were inoculated with a conidial suspension of P. longisetula (2 × 10⁵ conidia/ml), and leaf samples were collected at 48, 72, 96 and 144 h after inoculation (hai) for observation in the scanning electron microscope. Conidia germinated within 48 hai. At 72 hai, conidia had formed very long germ tubes over the epidermal cells without any evidence of appressorial formation nor direct penetration. At 96 hai, fungal hyphae grew inter‐ and intracellularly in the lacunous parenchyma and also through tracheary elements. Pycnidia were first observed on the leaf surface at 96 hai. At 144 hai, conidia of P. longisetula were first liberated from the pycnidia. This study adds new information to better understand of the infection process of P. longisetula that may help in developing more effective disease control strategies.     

Consecutive monitoring of lifelong production of conidia by individual conidiophores of Blumeria graminis f. sp. hordei on barley leaves by digital microscopic techniques with electrostatic micromanipulation

Conidial formation and secession by living conidiophores of Blumeria graminis f. sp. hordei on barley leaves were consecutively monitored using a high-fidelity digital microscopic technique combined with electrostatic micromanipulation to trap the released conidia. Conidial chains formed on conidiophores through a series of septum-mediated division and growth of generative cells. Apical conidial cells on the conidiophores were abstricted after the conidial chains developed ten conidial cells. The conidia were electrically conductive, and a positive charge was induced in the cells by a negatively polarized insulator probe (ebonite). The electrostatic force between the conidia and the insulator was used to attract the abstricted conidia from the conidiophores on leaves. This conidium movement from the targeted conidiophore to the rod was directly viewed under the digital microscope, and the length of the interval between conidial septation and secession, the total number of the conidia produced by a single conidiophore, and the modes of conidiogenesis were clarified. During the stage of conidial secession, the generative cells pushed new conidial cells upwards by repeated division and growth. The successive release of two apical conidia was synchronized with the successive septation and growth of a generative cell. The release ceased after 4-5 conidia were released without division and growth of the generative cell. Thus, the life of an individual conidiophore (from the erection of the conidiophore to the release of the final conidium) was shown to be 107 h and to produce an average of 33 conidia. To our knowledge, this is the first report on the direct estimation of life-long conidial production by a powdery mildew on host leaves.     

Antifeedant Properties of Destruxins and their Potential Use with the Entomogenous Fungus Metarhizium anisopliae for Improved Control of Crucifer Pests

Destruxins A, B and E, produced by the entomogenous fungus Metarhizium anisopliae, are insecticidal but comparatively low doses have antifeedant properties. Treatment of cabbage leaf discs with destruxins significantly reduced feeding by larvae of Plutella xylostella and Phaedon cochleariae in both choice and no-choice assays. The Antifeedant Index (AI) was dose related and there were significant differences between treated and untreated leaves. The AI and acute toxicity assays suggest that insect death was due to a combination of the starvation and toxicity effects of destruxins. In whole plant experiments, adults and larvae of P. cochleariae were found to be more susceptible to infection by M. anisopliae V245 if it was used in conjunction with a crude destruxin mixture. Destruxins drove larvae off the plant, irrespective of which leaf surface was treated. Adults could be forced to the adaxial or abaxial surface of leaves using the crude destruxin. Mortality was usually more consistent and generally greater if adults were forced to abaxial than adaxial surfaces inoculated with the fungus. High humidity on the abaxial surface favoured conidia germination and infection. Mortality was also greater for adults dusted with the pathogen and forced to the abaxial rather than to the adaxial leaf surface. The increased movement and starvation associated with destruxin treatment may also have stressed the insects making them more susceptible to infection.     

Morphological characterization and optimization of conditions for conidial production of Elsinoë ampelina,the causal organism of grapevine anthracnose

Grape anthracnose, which is caused by Elsinoë ampelina, is a disease that negatively affects grape production. This study aimed to investigate the effects of aeration, temperature, light, and preculture period on the formation of E. ampelina conidia and conidial germination and virulence. The colony morphology on potato dextrose agar (PDA) plates was more diverse than that in PDA bottles. The assessment of different culture methods, temperatures, light conditions, and preculture periods revealed that optimal conidial production occurred on 25‐day‐old colonies grown in PDA bottles at 21°C for 24 hr in the dark. The cultures in PDA bottles consistently produced approximately 5.0 × 10⁶ conidia under these conditions. No conidial formation occurred when the cultures were kept at 25°C in the dark. The highest germination rate of E. ampelina was 80% at 25°C after 24 hr, whereas no germination was observed at 17°C after 12 hr. Pathogenicity tests revealed that symptoms of the disease were observed 4 days postinoculation (dpi) on leaves of Vitis vinifera cv. Red Globe. New conidia were observed on the lesions at 8 dpi. This study provides an effective method for the conidial production of E. ampelina that may also be applicable for other Elsinoë fungal species.     

Ontogenetic changes in stomatal size and conductance of sunflowers

The ontogenetic changes in stomatal size, frequency and conductance (g_s) on abaxial and adaxial leaf surfaces of sunflower plants (Helianthus annuus L. Russian Mammoth) were examined under controlled environmental conditions. The stomatal frequency on the adaxial and abaxial leaf surfaces decreased with leaf ontogeny and insertion level. The ratio of adaxial to abaxial stomatal frequency did not change with leaf ontogeny and insertion level, and 42–44% of total stomata was apportioned to the adaxial surface. Ontogenetic changes in stomatal pore length were detected and increased with ontogenesis. The stomatal length of both leaf surfaces had linear relationships with leaf area. Ontogenetic changes in g_s were similar between the two surfaces. However the adaxial g_s was lower than abaxial g_s in leaves of higher insertion levels. Conductance had a linear relationship with width x frequency but not with pore area.     

Conidia of the tomato powdery mildew <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> initiate germ tubes at a predetermined site

The emergence of germ tubes from the conidia of powdery mildew fungi is the first morphological event of the infection process, preceding appressoria formation, peg penetration and primary haustoria formation. Germination patterns of the conidia are specific in powdery mildew fungi and therefore considered useful for identification. In the present study, we examined conidial germination of the tomato powdery mildew Oidium neolycopersici KTP-01 in order to clarify whether germ tube emergence site in KTP-01 conidia is determined by the first contact of the conidia to leaves (as found for the conidia of barley powdery mildew), or alternatively is predetermined and is unrelated to contact stimulus. Highly germinative conidia of KTP-01 were collected from conidial pseudochains on conidiophores in colonies on tomato leaves using two methods involving an electrostatic spore attractor and a blower. In the electrostatic spore attraction method, the conidia were attracted to the electrified insulator probe of the spore collector—this being the first contact stimulus for the conidia. In addition, the blowing method was used as a model of natural infection; pseudochain conidia were transferred to detached leaves by air (1 m/s) from a blower. Thus, landing on the leaves was the first contact for the conidia. Furthermore, conidia were also blown onto an artificial membrane (Parafilm-coated glass slides forming a hydrophobic surface) or solidified agar plates in Petri dishes (hydrophilic surface). Eventually, almost all conidia on the probe and on tomato leaves or artificial hydrophobic and hydrophilic surfaces synchronously germinated within 6 h of incubation, indicating that the first contact of the conidia with any of the aforementioned substrata was an effective germination induction signal. Germ tube emergence sites were exclusively subterminal on the conidia. Moreover, the germ tubes emerged without any relation to the sites touched first on the conidia. Thus, the present study strongly indicates that conidia of O. neolycopersici produce germ tubes at a predetermined site.     

Conidia of <Emphasis Type="Italic">Erysiphe trifoliorum</Emphasis> attempt penetration twice during a two-step germination process on non-host barley leaves and an artificial hydrophobic surface

In the present study, using a high-fidelity digital microscope, we observed the sequence of appressorial development on the germ tubes of a powdery mildew fungus isolated from red clover leaves. Based on its morphological characteristics and rDNA internal transcribed spacer (ITS) sequences, the fungus was identified as Erysiphe trifoliorum, and one of its isolates, designated as KRCP-4N, was used in this work. The conidial germination of isolate KRCP-4N was studied on host (red clover) and non-host (barley) leaves, as well as on an artificial hydrophobic membrane (Parafilm). More than 90% of conidia germinated synchronously and developed dichotomous appressoria (symmetrical double-headed appressoria) on all substrata used. On host leaves, all appressorium-forming conidia developed hyphae (colony-forming hyphae) from conidial bodies without extending germ tubes from the tips of the appressoria. On non-host leaves and on Parafilm-covered glass slides, however, all conidia extended germ tubes from one side of dichotomous appressoria (two-step germination). In addition to the dichotomous appressoria, we detected a few conidia that produced hooked appressoria and extended germ tubes from the tip of the appressorium. Penetration attempts by KRCP-4N conidia on barley leaves were impeded by papillae formed at penetration sites beneath these two types of appressorium. From these results, we conclude that the “two-step germination” of E. trifoliorum KRCP-4N conidia is the result of an unsuccessful penetration attempt, causing diversity in appressorial shape.     

Seasonal and diurnal leaf orientation,bifacial sunlight incidence,and leaf structure in the sand dune herb Hydrocotyle bonariensis

A conceptual model has been proposed whereby leaf orientation and resulting sunlight exposure dictate functional leaf structure. Specifically, the model states that this relationship is driven by the absolute amount and ratio of incident sunlight on adaxial and abaxial leaf surfaces. To test this model, the relationships between corresponding values of leaf orientation and incident sunlight on both leaf surfaces were measured for the sand dune herb Hydrocotyle bonariensis over a growth season, along with examination of leaf structure. For mature leaves, leaf angle from horizontal and azimuth angle significantly increased over the growing season, indicating diurnal midday avoidance and seasonal maximization of incident sunlight. Consequently, seasonal changes in leaf orientation resulted in an overall decrease in midday sunlight incidence on the adaxial surface and a slight shift in the daily occurrence of peak abaxial incidence. Adaxial surfaces received three to four times more sunlight than abaxial surfaces, and leaf cross-sections revealed relatively thick (564 μm) leaves with multiple adaxial palisade layers and stomata on both leaf surfaces, as predicted by the conceptual model and measured ratio of incident sunlight on adaxial and abaxial leaf surfaces. These data provide further evidence of the relationship between leaf orientation and resulting absolute levels of sunlight incidence on both leaf surfaces, as well as their ratio, and corresponding differences in internal and external leaf structure.     

Leaf surface preference in the cabbage worm,Pieris rapae crucivora,and parasitism by the gregarious parasitoid Cotesia glomerata

Leaf surface preference of the cabbage worm, Pieris rapae crucivora Boisduval (Lepidoptera: Pieridae), for cabbage, Brassica oleracea L. var. capitata (Brassicaceae), and parasitism by the parasitoid Cotesia glomerata (L.) (Hymenoptera: Braconidae) were investigated experimentally in the laboratory. Female butterflies did not discriminate between the adaxial and abaxial surfaces of cabbage leaves when laying eggs on a vertically placed leaf. Larvae also did not discriminate between the adaxial and abaxial surfaces throughout their larval life. However, second and third instars preferred the lower surface of horizontally placed leaves to the upper surface, irrespective of whether they had hatched on the upper or lower side; other instars showed no preference for the lower surface. Parasitism rates of first and second instars on the upper surface were higher than those of larvae on the lower surface. Egg distribution on leaf surfaces and the leaf surface preference by young larvae are discussed in terms of avoidance of parasitism by the parasitoid C. glomerata.     

Dorsoventral variations in dark chilling effects on photosynthesis and stomatal function in Paspalum dilatatum leaves

The effects of dark chilling on the leaf-side-specific regulation of photosynthesis were characterized in the C(4) grass Paspalum dilatatum. CO(2)- and light-response curves for photosynthesis and associated parameters were measured on whole leaves and on each leaf side independently under adaxial and abaxial illumination before and after plants were exposed to dark chilling for one or two consecutive nights. The stomata closed on the adaxial sides of the leaves under abaxial illumination and no CO(2) uptake could be detected on this surface. However, high rates of whole leaf photosynthesis were still observed because CO(2) assimilation rates were increased on the abaxial sides of the leaves under abaxial illumination. Under adaxial illumination both leaf surfaces contributed to the inhibition of whole leaf photosynthesis observed after one night of chilling. After two nights of chilling photosynthesis remained inhibited on the abaxial side of the leaf but the adaxial side had recovered, an effect related to increased maximal ribulose-1,5-bisphosphate carboxylation rates (V(cmax)) and enhanced maximal electron transport rates (J(max)). Under abaxial illumination, whole leaf photosynthesis was decreased only after the second night of chilling. The chilling-dependent inhibition of photosynthesis was located largely on the abaxial side of the leaf and was related to decreased V(cmax) and J(max), but not to the maximal phosphoenolpyruvate carboxylase carboxylation rate (V(pmax)). Each side of the leaf therefore exhibits a unique sensitivity to stress and recovery. Side-specific responses to stress are related to differences in the control of enzyme and photosynthetic electron transport activities.     

Powdery Mildew on Mulberry in Yunnan,China is Distinct from Other Phyllactinia spp. on Morus

The occurrence of an epidemic outbreak of a powdery mildew disease on mulberry in Yunnan province, China, is reported. Its symptoms are characteristic for powdery mildews and visible as white pathches covering the abaxial surfaces of leaves leading to chlorosis and necrosis. The pathogen is morphologically barely distinguishable from Phyllactinia moricola. However, it exhibits several new morphological characteristics which 2–3 conidia could be formed in short chains at the apex of the conidiophores and the conidia could produce two germ tubes in any position. Phylogenetic analyses of ITS sequences show that the pathogen has a close genetic relationship with P. moricola and Ph. broussonetiae‐kaempferi, two species on hosts belonging to family Moraceae. However, the ITS differences between Japanese sequences and the Chinese sequence derived from mulberry are greater than expected for a single species and suggest a cryptic species in China, but the present data are not sufficient for a final conclusion. Therefore, the Morus powdery mildew in Yunnan can currently only be classified as Phyllactinia sp. Morphological features, including conidial germination pattern of this powdery mildew are described in detail, and the local climatic conditions of the disease are analysed, which will provide the base for finding an effective method, including bio‐control, to control the disease under local conditions.     

Inclination of sun and shade leaves influences chloroplast light harvesting and utilization

Light harvesting and utilization by chloroplasts located near the adaxial vs the abaxial surface of sun and shade leaves were examined by fluorometry in two herbaceous perennials that differed in their anatomy and leaf inclination. Leaves of Thermopsis montana had well-developed palisade and spongy mesophyll whereas the photosynthetic tissue of Smilacina stellata consisted of spongy mesophyll only. Leaf orientation depended upon the irradiance during leaf development. When grown under low-light levels, leaves of S. stellata and T. montana were nearly horizontal, whereas under high-light levels, S. stellata leaves and T. montana leaves were inclined 60⁰ and 30⁰, respectively. Leaf inclination increased the amount of light that was intercepted by the lower leaf surfaces and affected the photosynthetic properties of the chloroplasts located near the abaxial leaf surface. The slowest rates of quinone pool reduction and reoxidation were found in chloroplasts located near the adaxial leaf surface of T. montana plants grown under high light, indicating large quinone pools in these chloroplasts. Chloroplasts near the abaxial surface of low-light leaves had lower light utilization capacities as shown by photochemical quenching measurements. The amount of photosystem II (PSII) down regulation, measured from each leaf surface, was also found to be influenced by irradiance and leaf inclination. The greatest difference between down regulation monitored from the adaxial vs abaxial surfaces was found in plants with horizontal leaves. Different energy dissipation mechanisms may be employed by the two species. Values for down regulation in S. stellata were 2–3 times higher than those in T. montana, while the portion of the PSII population which was found to be Q_B nonreducing was 4–6 times lower in high light S. stellata leaves than in T. montana. All values of Stern-Volmer type nonphotochemical quenching (NPQ) from S. stellata leaves were similar when quenching analysis was performed at actinic irradiances that were higher than the irradiance to which the leaf surface was exposed during growth. In contrast, with T. montana, NPQ values from the abaxial leaf surface were up to 45% higher than those from the adaxial leaf surface regardless of growth conditions. The observed differences in chloroplast properties between species and between the adaxial and abaxial leaf surfaces may depend upon a complex interaction among light, leaf anatomy and leaf inclination.     

Interactions of colonisation density and leaf environments on survival of Trioza eugeniae nymphs

1. Female eugenia psyllids Trioza eugeniae oviposit on the margins of expanding young Syzygium paniculatum leaves. The developing nymphs, feeding within pit‐shaped galls on the leaves, cause the leaves to become curled and deformed. The degree of leaf curling was correlated positively with densities of T. eugeniae nymphs. 2. High relative humidity increased persistence of nymphs on leaves at low insect densities, but persistence did not differ between high or low relative humidity conditions when nymphal densities were high and leaves were greatly curled. 3. Direct insolation increased nymphal mortality. Nymphs on the abaxial leaf surface in the direct sun had lower mortality than similarly exposed nymphs on the adaxial leaf surface. 4. Field populations showed high preference for abaxial leaf surfaces and a stronger preference for shaded adaxial surfaces than for exposed adaxial surfaces. 5. Adverse environmental conditions of direct insolation and low relative humidity may be mitigated by leaf curl associated with moderate populations, however competition at high nymphal density supersedes any potential benefit arising from leaf curling and has a negative effect on nymphal survival.     

Influence of microclimate on Drechslera leaf spot of young coconuts: effect of desiccation on spore survival and of moisture and shade on infection and disease development

Culture-produced conidia of Drechslera incurvata from coconut failed to germinate on the leaves of coconut seedlings incubating under the dry conditions of a greenhouse. Viability and rate of appressorium formation of artificially-dispersed, culture-produced conidia fell significantly during extended incubation of inoculated seedlings in the greenhouse, when 43% of conidia germinated after 90 days incubation compared with 62% at 59 days and 90% at 24 days. Field-produced conidia on excised leaves also lost viability upon storage in situ on the laboratory bench; germination fell from 60% at 3 months storage to 0×5% at 5 months and no germination at 6 months. Shading of seedlings in the field with saran cloth producing 30% shade or 50% shade depressed the amount of dew forming on leaves of young coconuts and significantly reduced both the number of infections from artificial inoculations and the severity of leaf spot disease developing subsequently.     

Evidence of Alternaria alternata Causing Leaf Spot of Aloe vera in Iran

A leaf spot and leaf blight disease was observed on Aloe vera plants as small, circular to oval dark brown necrotic sunken spots on leaves. Infected tissues collected from different sites in diseased fields were cultured on potato carrot agar medium, and the pathogen was identified as Alternaria alternata on the basis of morphological and cultural characteristics. The conidiophores were branched, straight, golden brown, smooth‐walled, measuring up to μm long by 3 μm wide with one conidial scar. The conidia were golden brown in colour and produced in long branched chains, obclavate in shape and in short conical flask. Pathogenicity tests conducted on healthy potted aloe plants in a glasshouse showed typical leaf spot symptoms after 4–7 days. The optimal temperature for the growth of A. alternata was 25°C.