The rates of fungal development and lesion formation in leaves of Vicia faba during infection by Botrytis cinerea and Botrytis fabae

Loss of the water droplet above inoculation sites during the first day after inoculation inhibited lesion formation by Botrytis cinerea and prevented the development of spreading lesions of B. fabae. With droplets present two general patterns of infection by B. cinerea were determined; in one, few or no symptoms were produced and in the other, limited lesions developed with marked browning of the inoculation site. Where few or no symptoms were produced, germination and germ-tube growth were inhibited on the leaf surface. B. cinerea was inhibited within the leaf at sites bearing limited lesions; invading hyphae were restricted to brown epidermal cells. Fungal growth on the leaf surface was greatest at sites with most browning beneath the droplet area. Variation in lesion development by B. cinerea could not be related to droplet position or leaf damage during normal preparation for inoculation. Plants differed in their susceptibility to lesion formation by B. cinerea. B. fabae, with droplet present, was not inhibited on the leaf surface and spread inter- and intra-cellularly beneath the inoculum drop and then into surrounding tissues. Delay in spread until the inoculation site was completely necrotic and colonized suggested that B. fabae is partially inhibited during the initial phase of infection. The rate of lesion spread varied in different plants and was most rapid in the youngest leaves.     

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.     

Root-colonization ability of antagonistic Streptomyces griseoviridis

Root-colonization ability of Streptomyces griseoviridis was tested on turnip rape (Brassica rapa subsp. oleifera) and carrot (Daucus carota) by the plate test and the sand-tube method. In the plate test, colonized root length of total root length was highly significantly greater for turnip rape roots (72%) from those for carrot roots (1%). In the sand-tube method, root-colonization ability was examined in nonsterile soil, and no water was added after sowing. Seeds were treated with spores of S. griseoviridis or the biofungicide Mycostop. Roots were cut into 2-cm segments, and the root segments and the rhizosphere soil were studied separately. Root-colonization frequencies and population densities of the microbe in the rhizosphere soil indicated that S. griseoviridis successfully colonized turnip rape but weakly colonized carrot. Root-colonization of turnip rape is accounted for as proliferation of S. griseoviridis in the rhizosphere of turnip rape seedlings and is not due to the movement of microbe through the rhizosphere by water infiltration.     

Short feeding period of carrot psyllid (Trioza apicalis) females at early growth stages of carrot reduces yield and causes leaf discolouration

Overwintered adult carrot psyllids [Trioza apicalis Förster (Homoptera: Psylloidea: Triozidae)] damage carrot [(Daucus carota ssp. sativum L.) (Apiaceae)] seedlings by phloem feeding on the leaves. The aim of this study was to investigate the carrot root and shoot growth in relation to carrot psyllid density during early growth stages. One, two, or three carrot psyllids were allowed to feed on carrot seedlings for 3 days. Leaf damage was measured at the 8‐leaf stage, and root, leaf fresh weight, and number of true leaves were measured at harvest. Both the age of the carrot seedling at infestation and the psyllid density had a significant effect on leaf damage at the 8‐leaf stage: seedlings damaged at the cotyledon stage exhibited more leaf damage than seedlings damaged at the 1‐leaf stage. A higher psyllid density significantly reduced the carrot root weight at harvest. The significant interaction of psyllid density with seedling age indicates that differently aged carrot seedlings responded differently to feeding: one psyllid feeding for 3 days at the cotyledon stage caused a significant yield loss, whereas three psyllids were needed to cause the same impact at the 1‐leaf stage. Carrot leaf weight at harvest was not reduced by carrot psyllid feeding: leaves recovered from the damage but roots did not. Our results confirm the farmers’ observations that a trap replacement period of 1 week for carrot psyllid monitoring is too long, especially at the cotyledon stage. Severe leaf discolouration on damaged carrots was observed at harvest. The possible reasons for this discolouration, such as toxin excreted in psyllid saliva or plant pathogenic mycoplasma infection, are discussed.     

The Association of Discula destructiva (Red.) Hyphae With Cornus florida (L.) Trichomes

Detached, surface-dismfested flowering dogwood (Cornus florida L.) leaves were inoculated with a Discula destructiva (Red.) spore suspension in an effort to identify inoculation and pre-penetration phenomena in the dogwood anthracnose pathosystem. Scanning electron micrographs show an association of D. destructiva hyphae with trichomes on the flowering dogwood upper leaf surface. The basal area of these leaf hairs may provide an entry point for Discula spp. colonization of and penetration into the dogwood leaf; however, the possible significance of this area as a unique ecological niche on the dogwood phylloplane should not be ignored.     

Pyruvate Kinase Activity in Crude Extracts of Leaves of Cynodon dactylonand Other C4Plants

A new extraction procedure and an LDH-coupled assay method are presented for the study of pyruvate kinase (PK) in leaf crude extracts from Cynodon dactylon(L.) Pers and other C₄plants. Extraction at pH 6.8 and assay at pH 6.2 facilitated the measuring of PK activity by eliminating phosphoenolpyruvate carboxylase interference more effectively than the thermal inactivation or chemical inhibition previously used. The method suggested did not affect the kinetic properties of PK as compared to the purified enzyme from C. dactylon.     

Effect of ethylene on auxin transport and metabolism in midrib sections in relation to leaf abscission of woody plants

Abstract The relationship between ethylene-induced leaf abscission and ethylene-induced inhibition of auxin transport in midrib sections of the leaf blade of Citrus sinensis L. Osbeck, Populus deltoides Bart, and Eucalyptus camaldulensis Dehn. was studied. These species differed greatly in their abscission response to ethylene. The kinetic trend of abscission resembled that of the inhibition of auxin transport in all three species. It is suggested that one of the main actions of ethylene in the leaf blade is to inhibit auxin transport in the veinal tissues, thus reducing the amount of auxin transported from the leaf blade to the abscission zone. Ethylene inhibited transport of both IAA (indole-3-acetic acid) and NAA (α-naphthaleneacetic acid) in the midrib sections. However, while ethylene enhanced the conjugation of IAA with aspartic acid and glucose in the apical (absorbing) segment of the midrib sections, it had little effect on the conjugation of NAA. The data indicate that auxin destruction through conjugation does not play a major role in the inhibition of auxin transport by ethylene.     

Antifeedant effects of in vitro culture extracts of the neem tree,Azadirachta indica against the desert locust (Schistocerca gregaria (Forskål))

Callus and micropropagated shoots were initiated from leaf explants of the neem tree, Azadirachta indica A. Juss. A variety of whole plant and in vitro cell cultures from neem seedlings of Ghanian origin were tested for insect antifeedant compounds using the desert locust (Schistocerca gregaria (Forskål)). Feeding suppression occurred when whole extracts of seed, leaf, callus, suspension and shoot cultures were tested in no-choice feeding bioassays. Controls of sucrose, carrot callus and the plant growth medium showed no feeding deterrence. Azadirachtin, the main known antifeedant in neem seed kernels, was quantified from a seed extract by HPLC but was not detected in any of the other extracts. Antifeedancy was determined during batch growth of a suspension culture which had been in culture for 5 months; results indicated that antifeedants were still being formed and that levels increased after maximum biomass was attained.     

Regeneration from the Leaf of Atrichum undulatum (Hedw.) P. Beauv.

Abstract

Regeneration of new plants from the leaves of Atrichum undulatum will take place after 6 weeks if they are placed with the abaxial surface in contact with damp filter-paper in a saturated atmosphere.

The new plants arise from the surface cells of the midrib area of the leaf where there is a discontinuity of the photosynthetic filaments. This occurs mainly in the distal third of the leaf.

As the leaf ages it loses it capacity for regeneration. There are two regeneration processes, one which occurs when the leaf is separated from the stem and the only wound is at the point of attachment to the stem, and the second when other wounds are inflicted on the leaf. Regeneration due to wounding is greatest in the distal half of the leaf, mutilations near the base of the leaf having less effect on the percentage of regenerated plants.

There seems to be a potential for regeneration (both normal and as a result of wounding) which is greatest in the distal third of the leaf and decreases towards the base of the leaf of A. undulatum. This is compared with the leaf of Physcomitrium turbinatum, and the greater complexity of the process in the former is considered to be the result of greater differentiation of the leaf.     

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.     

Effects of carrot psyllid (Trioza apicalis) feeding on carrot yield and content of sugars and phenolic compounds

Carrot psyllid, Trioza apicalis, is a serious pest of carrot in Northern Europe, as it can significantly damage young carrot seedlings in a period as short as 3 days. This study was conducted to investigate effects of carrot psyllid feeding at different plant growth stages on carrot yield and to assess changes in content of sugars, phenolics and related compounds in carrot roots resulting from the psyllid feeding. In addition, reflectance of carrot leaves was measured to assess the intensity of discolouration in damaged leaves. Results showed that carrot yield was significantly reduced by a 3‐day carrot psyllid feeding period when the seedlings were exposed to psyllids at 1‐ or 2‐leaf stage. However, at 4‐leaf stage feeding by one carrot psyllid did not reduce yield. Sucrose concentration in the damaged roots was significantly decreased, whereas concentrations of some phenolic compounds were significantly increased. The reflectance of leaves of damaged carrots differed significantly from those of undamaged control leaves. These observations indicate that carrot psyllid damage has potential to lower not only the carrot yield, but also the carrot crop quality. No phytoplasma was detected in the carrots exposed to psyllids, but recently, T. apicalis has been associated with ‘Candidatus Liberibacter solanacearum’. The role of carrot psyllid feeding and the psyllid‐associated bacterium in the damage formation are discussed.     

Surface Ultrastructural Studies on Ingress and Establishment of Pseudomonas syringae pv. mori on Mulberry Leaves

P. syringae pv. mori multiplied on leaf surface and colonized particularly on the cystoliths and in the grooves of veins. The masses of bacteria were associated with necrotic spots, which appeared 9 days after inoculation. The studies also revealed that the bacterium invaded leaf tissues through cystoliths. However, it did not enter through stomata and trichomes which had commonly been observed in most of the plant pathogenic bacteria.     

Bud formation in leaves,leaf fragments and midrib pieces of Heloniopsis orientalis (Liliaceae)

Summary Isolated leaves, leaf fragments and pieces of the midrib portion devoid of lamina, of Heloniopsis orientalis were grown on an inorganic nutrient medium without organic nutrients and growth regulators in order to investigate their regenerative ability. Bud formation in intact, attached leaves occurs only at the tip, in isolated leaves at the tip and the base, whereas leaf fragments cut transversely at a distance from the tip and isolated midrib pieces form numerous shoot buds in a random distribution. Lamina fragments lacking midrib frequently fail to regenerate even after a long time of culture. It is suggested that endogeneous growth regulators in the leaf, especially the vascular tissues, play an important role in bud initiation. Very young leaves of Heloniopsis are capable forming buds and roots when isolated from the mother plants.     

中国瓜馥木属植物叶片结构的解剖观察

采用叶片表皮离析法、扫描电镜和石蜡制片法,研究了中国分布的番荔枝科瓜馥木属19种植物叶片的形态结构。结果表明:瓜馥木属叶片形态和结构具有较多的相似性,如叶片表皮均具有2-多个细胞的单列丝状毛,表皮细胞内具有一个晶簇,气孔器均为平列型,只分布在叶片的远轴面,远轴面的表皮细胞平周壁形成一个乳突,叶片主脉的维管组织除具有正常的维管组织外,还具有一个小的副维管束等,这些特征有助于区分番荔枝科植物的属间关系,支持瓜馥木属是一个很自然的类群。但叶片表皮毛的形态及分布,表皮细胞的形状、叶肉中栅栏组织和海绵组织的结构、远轴面的乳突大小以及叶主脉维管组织的细微结构则具有种间差异。尤其有助于区分小萼瓜馥木和黑风藤、广西瓜馥木和独山瓜馥木、上思瓜馥木和东方瓜馥木等形态相似、难以区分的植物种类。该研究结果为该属的系统研究和种间正确区分提供了重要资料。     

Isolation of endophytic fungi from leaves of Pasania edulis and their within-leaf distributions

 Endophytic fungi were isolated from leaves of Pasania edulis, one of the most important trees of the warm temperate forests in southern Kyushu, by the surface sterilization method using H₂O₂ as a sterilizing agent. From a tree in the Experimental Nursery of Kagoshima University, located at the city of Kagoshima, Phyllosticta sp. and Colletotrichum spp. were frequently isolated. From a stand in a laurel forest in Mt. Takakuma, an ascomycetous fungus (Ascomycete sp. 1) and Phomopsis sp. were frequently isolated. Phyllosticta sp. was isolated more frequently from petiole segments and leaf segments with midrib and Phomopsis sp. from petiole segments and leaf-base segments with midrib than other segments. Colletotrichum spp. were isolated less frequently from petioles and Ascomycete sp. 1 from petiole segments and leaf-base segments with midrib than other segments. As possible causes of such biases in within-leaf distributions of the endophytes, differences in infection modes and negative interactions of major endophytes within leaves are suggested. Received: December 13, 2001 / Accepted: June 7, 2002 Acknowledgments The authors thank the staff members of the Experimental Forests of Kagoshima University for enabling the present study. Correspondence to:K. Hata     

Estimation of genetic diversity among Turkish kale populations (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">acephala</Emphasis> L.) using RAPD markers

Twenty populations of kale (Brassica oleracea var. acephala L.) selected from 127 populations in terms of yield and leaf quality characteristics as superior types using weight-based ranking method from the Black Sea Region of Turkey were evaluated at the DNA level using randomly amplified polymorphic DNA (RAPD) markers compared to some morphological characters. The seven primers selected from 100 decamers used generated 110 bands, of which 60 (54.5%) were polymorphic. Jaccard’s genetic distances were calculated and dendrogram was generated using the UPGMA algorithm. The dendrogram obtained was classified into three main groups and four subgroups. The accessions showed a limited clustering as compared to morphological characters such as the number of leaves, intentation of the leaf margin, leaf and midrib color, and thickness of midrib, than geographical characteristics. Leaf color and midrib thickness characters clustered in the same group as OR49 and G18 accessions; S20, G6, and OR37 accessions, respectively. The text was submitted by the authors in English.     

Plant–Microbe Interactions: Wetting of Ivy (Hedera helix L.) Leaf Surfaces in Relation to Colonization by Epiphytic Microorganisms

Abstract Leaf wettability, cuticular wax composition, and microbial colonization of upper and lower leaf surfaces of ivy (Hedera helix L.) was investigated for young and old leaves sampled in June and September. Contact angles of aqueous buffered solutions measured on young leaf surfaces ranged between 76° and 86° and were not dependent on the pH value of the applied droplets. Contact angles measured on old leaf surfaces were up to 32°, significantly lower than on young leaf surfaces. Furthermore, contact angles were significantly lower using aqueous solutions of pH 9.0 compared to pH 3.0, indicating the influence of ionizable functional groups on leaf surface wetting properties. Observed changes in leaf wetting properties did not correlate with different levels of alkanoic acids in cuticular waxes. However, microscopic examination of the leaf surfaces indicated the influence of epiphytic microorganisms on wetting properties of old leaves, since their surfaces were always colonized by epiphytic microorganisms (filamentous fungi, yeasts, and bacteria), whereas surfaces of young leaves were basically clean. In order to analyze the effect of epiphytic microorganisms on leaf surface wetting, surfaces of young and clean ivy leaves were artificially colonized with Pseudomonas fluorescens. This resulted in a significant increase and a pH dependence of leaf surface wetting in the same way as it was observed on old ivy leaf surfaces. From these results it can be deduced that the native wetting properties of leaf surfaces can be significantly masked by the presence of epiphytic microorganisms. The ecological implications of altered wetting properties for microorganisms using the leaf/atmosphere interface as habitat are discussed. Received: 20 March 1999; Accepted: 5 July 1999; Online Publication: 18 July 2000     

Co-ordinated development of the leaf midrib xylem with the lamina in Nicotiana tabacum

Background and Aims

The water-transport capacity of leaf venation is positively related to the leaf-lamina area, because the number and diameter of vein-xylem conduits are controlled to match the lamina area. This study aimed to investigate how this co-ordinated relationship between the leaf-lamina area and vein-xylem characteristics is achieved by examining the midrib xylem of tobacco leaves.

Methods

The changes in the midrib-xylem characteristics over time were quantified using leaves with four different final lamina areas. The measured data were fitted to sigmoidal functions. From the constants of the fitted curves, the final values in mature leaves, maximal developmental rates (V_Dev) and developmental duration (T_Dev) were estimated for each of the xylem characteristics. Whether it is the lamina or the midrib xylem that drives the co-ordinated development was examined by lamina removal from unfolding leaves. The effects of the application of 0·1 % IAA (indole-3-acetic acid) to leaves with the laminas removed were also analysed.

Key Results

For both the leaf lamina and the midrib-xylem characteristics, the differences in final values among leaves with different lamina areas were more strongly associated with those in V_Dev. Notably, the V_Dev values of the midrib-xylem characteristics were related to those of the leaf-lamina area. By lamina removal, the conduit diameter was reduced but the number of conduits did not significantly change. By IAA application, the decrease in the conduit diameter was halted, and the number of conduits in the midrib xylem increased.

Conclusions

According to the results, the V_Dev values of the lamina area and the midrib-xylem characteristics changed in a co-ordinated manner, so that the water-transport capacity of the midrib xylem was positively related to the leaf-lamina area. The results also suggest that IAA derived from the leaf lamina plays a crucial role in the development of the leaf venation.     

Quantification of plant surface metabolites by matrix‐assisted laser desorption–ionization mass spectrometry imaging: glucosinolates on Arabidopsis thaliana leaves

The localization of metabolites on plant surfaces has been problematic because of the limitations of current methodologies. Attempts to localize glucosinolates, the sulfur‐rich defense compounds of the order Brassicales, on leaf surfaces have given many contradictory results depending on the method employed. Here we developed a matrix‐assisted laser desorption–ionization (MALDI) mass spectrometry protocol to detect surface glucosinolates on Arabidopsis thaliana leaves by applying the MALDI matrix through sublimation. Quantification was accomplished by spotting glucosinolate standards directly on the leaf surface. The A. thaliana leaf surface was found to contain approximately 15 nmol of total glucosinolate per leaf with about 50 pmol mm^?2 on abaxial (bottom) surfaces and 15–30 times less on adaxial (top) surfaces. Of the major compounds detected, 4‐methylsulfinylbutylglucosinolate, indol‐3‐ylmethylglucosinolate, and 8‐methylsulfinyloctylglucosinolate were also major components of the leaf interior, but the second most abundant glucosinolate on the surface, 4‐methylthiobutylglucosinolate, was only a trace component of the interior. Distribution on the surface was relatively uniform in contrast to the interior, where glucosinolates were distributed more abundantly in the midrib and periphery than the rest of the leaf. These results were confirmed by two other mass spectrometry‐based techniques, laser ablation electrospray ionization and liquid extraction surface analysis. The concentrations of glucosinolates on A. thaliana leaf surfaces were found to be sufficient to attract the specialist feeding lepidopterans Plutella xylostella and Pieris rapae for oviposition. The methods employed here should be easily applied to other plant species and metabolites.