The infection process, sporulation and survival of Alternaria helianthi on sunflower

Electron microscopy was used to study the infection of sunflower leaves by Alternaria helianthi. Conidia germinated by producing one to many germ tubes which grew across the leaf surface before forming appressoria. The fungus directly penetrated its host through the cuticle and epidermis. Entry into the host through wounds and stomates was also observed. Extracellular sheaths were found to be associated with germ tubes and intercellular hyphae of A. helianthi. Conidiophores developed through collapsed stomates, from leaf veins, trichomes and also from mycelium growing across the host leaf surface. Microcylic conidia were produced directly from parent conidia under certain conditions. Studies using a volumetric spore trap showed that the airborne spore concentration followed a distinct periodicity with peaks occurring between 0900 and 1100 h each day. Laboratory studies showed that safflower, noogoora burr and bathurst burr could serve as alternative hosts for A. helianthi. The pathogen was readily isolated from sunflower crop debris from a diseased crop that had been harvested 1 yr earlier.     

Cytology of induced systemic resistance of cucumber to Colletotrichum lagenarium

The infection of cucumber leaves by Colletotrichum lagenarium was studied using cytological methods. Its progress in untreated plants was compared with that in plants in which systemic resistance had been induced by pre-infecting the first true leaf with the same fungus. In induced plants, a reduction of fungal development was observed at the leaf surface, in the epidermis, and in the mesophyll. On the leaf surface, formation of appressoria was slightly reduced. In the epidermis, enhanced formation of papillae beneath appressoria, and possibly increased lignification of entire cells, correlated with reduced development of infection hyphae. Papillae contained callose, identified by staining with aniline-blue fluorochrome and digestion with -1,3-glucanase, as a main structural component. In the mesophyll, reduced fungal development provided evidence for the existence of an additional induced defence reaction. The results imply that preinfection elicited a systemic, multicomponent defence reaction of the host plant against the fungus.Dedicated to the memory of Professor H. Grisebach     

Surface ultrastructure of the uredinial stage ofCerotelium fici and its infection process on mulberry

Surface morphology of uredinia and urediniospores ofCerotelium fici (Cast.) Arth., and its infection process in mulberry (Morus alba L.) have been described using the scanning electron microscope. The uredinia ofC. fici are paraphysate and bear pedicellate urediniospores. The surface morphology of urediniospore is similar to most of the rust fungi which have pedicellate urediniospores. The infection process ofC. fici on mulberry leaves differs from other rust fungi in not forming appressoria over the stomates. Further, the germ tube of the urediniospore crosses over the stomata, and sometimes forms an appressorium close to the stoma rather than forming over it. Thus, the present study indicates that the formation of appressoria byC. fici on mulberry leaves is not site specific but an independent, specialized and inherent mechanism required byC. fici to penetrate the mulberry leaf cuticle and epidermis.     

Involvement of carbohydrates and cytokinins in pathogenicity ofHelminthosporium carbonum

Significant stimulation of the number of appressoria, penetration and colonization by conidia ofHelminthosporium carbonum occurred on decolorized maize leaves when exogenous carbohydrates and leaf leachates were added. Germination and germ tube length, however, did not exhibit appreciable differences on decolorized or non-decolorized maize leaves. Lower germination was recorded by leached conidia on decolorized leaves; while appressoria, penetration and colonization were absent. Addition of exogenous nutrients (sucrose>leaf leachates>yeast extract>glucose) enabled conidia to accomplish appressoria, penetration and colonization. Optimum levels for various nutrients observed were 2% (w/v) sucrose/glucose or 0.1% (w/v) yeast extract. Higher concentrations inhibited the infection stages of the pathogen. Depletion of host carbohydrates from green islands/infection sites adversely affect appressoria formation, penetration and colonization; and the loss of carbohydrates from the spore affects germination. Cytokinin-like activity at the infection site/green islands increased with the period of incubation of the host as compared to the surrounding tissue or tissue under water drops. The culture filtrate extracts ofH. carbonum recorded cytokinin-like activity which increased with growth of the fungus. TLC (thin layer chromatography) of cytokinin-like substances (tissue extract and culture filtrate) revealed major activity was confined to Rf zones 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. These substances increase at infection sites by virtue of which carbohydrates accumulate at these sites ensuring a continuous supply to the growing pathogen.     

Cytology of induced systemic resistance of tomato to Phytophthora infestans

The infection of tomato leaves by Phytophthora infestans was followed using cytological methods. Fungal ingress and plant reactions in untreated and induced resistant plants were studied. Systemic disease resistance was induced by a local pre-infection with the same fungus. Induction retarded fungal progress at the leaf surface, epidermis and in the mesophyll. The reduced numbers of germinated cysts indicate the presence of fungitoxic substances on the leaf surface of induced plants. Frequency of fungal penetration through the outer epidermal cell wall was reduced, but only in plants exhibiting a high level of induced resistance. Autofluor-escent material, indicating the presence of lignin-like substances, accumulated rapidly beneath some of the appressoria, but this plant response was similar in induced and non-induced plants. Staining with aniline blue indicated that callose deposition was not involved in induced resistance. Thus, none of the cytologically investigated plant reactions correlated with the reduced penetration frequency observed. In the mesophyll, however, the cytological picture corresponding to a hypersensitive reaction occurred more often in induced plants. It is concluded that reduction of disease severity by induction is the result of the combined action of several successive defence reactions.Dedicated to the memory of Professor H. Grisebach     

PR-1 protein inhibits the differentiation of rust infection hyphae in leaves of acquired resistant broad bean

Treatment of broad bean leaves with salicylic acid (SA) or 2, 6-dichloro-isonicotinic acid (DCINA) induces resistance against the rust fungus Uromyces fabae resulting in reduced rust pustule density. Light-microscopy studies showed that in induced resistant plants the rust fungus is inhibited immediately after penetration through the stomatal pore. The differentiation of infection structures growing within the intercellular space of the leaf, i.e. infection hyphae and haustorial mother cells, is inhibited. Furthermore, low-temperature scanning electron microscopy studies of freeze fractures revealed protrusions at the tips of infection hyphae growing in induced resistant broad bean leaves. Treatment of in vitro-differentiating rust infection structures with intercellular fluids (IFs) from induced resistant plants confirmed that the fungus is sensitive towards an apoplastic anti-fungal activity only after having formed appressoria. Other legume rusts such as U. vignae and U. appendiculatus were likewise inhibited in the presence of IF from SA-treated broad bean leaves. Heterologous antibodies were used to study changes in the extracellular pathogenesis-related (PR) protein pattern after resistance induction. Western blots indicated that chitinases and beta-1,3-glucanases were present in both induced and control plants. In contrast, PR-1 proteins were newly synthesized in response to SA or DCINA application. The major induced PR-1 protein was purified and exhibited strong differentiation-inhibiting activity towards U. fabae infection structures. We conclude that the inhibition of rust infection hyphae in acquired resistant broad bean plants is mainly due to the anti-fungal activity of this induced basic PR-1 protein.     

A neighboring-inhibition model for stomate patterning

The patternization of stomate distribution was investigated in the first leaf of the sporophyte of the fern Dryopteris thelypteris and in the leaves of the jade plant Crassula argentae. In the fern leaf, stomates arise over a period of 1.1 days (26.4 hr) and attain a frequency of 0.186 of the epidermal cells while in the jade plant the formative period is over only 0.8 days (19.2 hr) and stomates reach a frequency of 0.090 of the epidermal cells. A computer model was devised to simulate the appearance of stomates by an induction process for a new stomate which then inhibits contiguous cells from becoming stomates. The validity of the model was demonstrated in that it gave values for stomate and stomate cluster frequencies upon calculating the frequency of free cells. For the fern leaf, the model required 57 iteration intervals, six iterations for the time from stomate induction until the adjacent cells were inhibited (a period of induction plus inhibitions), and an induction rate of 0.02 cells per time interval. From these theoretical values and the measured period of 26.4 hr when stomates arise during leaf development, a period of induction/inhibitions is calculated as 2.7 hr. In the model for the jade plant, 81 iteration intervals are required with six iterations per period of induction/inhibitions along with an induction rate of 0.002 cells per interation interval. These values give the duration for a period of induction/inhibitions in jade of 1.4 hr. This study describes the patterning process of stomate formation by an explicit, mathematical algorithm, and, from measurements of actual leaves, the various periods in the model can be assigned real time values.     

Morphogenic Regulation of Pathotoxin Synthesis in Cochliobolus carbonum

The fungus Cochliobolus carbonum causes leaf spot disease of maize. Highly virulent isolates of the pathogen produce a host-selective, peptide toxin that is active against susceptible genotypes of maize. Prior to infection, spores must germinate and differentiate appressoria, structures specialized for leaf penetration. Analysis of spore germination fluids by plasma desorption mass spectrometry, which allowed detection of as little as 0.5 ng toxin, revealed that spores induced to form appressoria in vitro synthesized and released the toxin at a time coincident with maturation of appressoria. Spores incubated under conditions that did not induce appressorium formation failed to produce toxin. These observations indicate that synthesis of the host-selective toxin, which is essential for successful pathogenesis of maize by C. carbonum, is regulated by infection-related morphogenesis.     

Does water and phosphorus uptake limit leaf growth of Rhizoctonia-infected wheat seedlings?

Wheat seedlings infected with a pure inoculum of the root-rotting fungus Rhizoctonia solani were grown in pots designed to fit in pressure chambers, to allow the effects of the Rhizoctonia infection on leaf growth to be studied while maintaining the leaves at elevated water status. Wheat was grown to the third leaf stage in soil inoculated with three different levels of Rhizoctonia, and the pots were then pressurised for seven days to maintain the leaf xylem at the point of bleeding (ie. the leaves were at full turgor). The reduction in leaf expansion caused by Rhizoctonia was not overcome by pressurisation, indicating that a reduced supply of water to the leaves was not responsible for reduced leaf growth. The addition of phosphorus to pots marginally deficient in P did not increase the leaf growth of Rhizoctonia-infected plants, despite increased P uptake to the leaves. These results indicate that a reduced supply of water to the leaves and a supply of phosphorus that was bordering on deficient was not the cause of the growth reduction in seedlings with Rhizoctonia infection. The nature of this reduced growth remains uncertain but may involve growth regulators produced by the fungus, or by the plant as a result of the infection process. The mechanism of these growth reductions is of interest as it may provide a key to the development of plant resistance mechanisms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Methanol Emission from Leaves (Enzymatic Detection of Gas-Phase Methanol and Relation of Methanol Fluxes to Stomatal Conductance and Leaf Development)

We recently reported the detection of methanol emissions from leaves (R. MacDonald, R. Fall [1993] Atmos Environ 27A: 1709-1713). This could represent a substantial flux of methanol to the atmosphere. Leaf methanol production and emission have not been investigated in detail, in part because of difficulties in sampling and analyzing methanol. In this study we used an enzymatic method to convert methanol to a fluorescent product and verified that leaves from several species emit methanol. Methanol was emitted almost exclusively from the abaxial surfaces of hypostomatous leaves but from both surfaces of amphistomatous leaves, suggesting that methanol exits leaves via stomates. The role of stomatal conductance was verified in experiments in which stomates were induced to close, resulting in reduced methanol. Free methanol was detected in bean leaf extracts, ranging from 26.8 [mu]g g-1 fresh weight in young leaves to 10.0 [mu]g g-1 fresh weight in older leaves. Methanol emission was related to leaf development, generally declining with increasing leaf age after leaf expansion; this is consistent with volatilization from a cellular pool that declines in older leaves. It is possible that leaf emission could be a major source of methanol found in the atmosphere of forests.     

Infection and Stroma Formation by Venturia inaequalis on Apple Leaves with Different Degrees of Susceptibility to Scab

Conidia of Venturia inaequalis germinated and formed appressoria on all leaves independently of age and origin within the first 15 hours after inoculation. On the youngest expanded leaves of the apple variety Golden Delicious 80% of the conidia developed stromata within the first 24 hours, sporulation occurred after 8 days. On the second leaf (counted from the top of the twig) stroma formation was slowed down compared to the first and sporulation occurred after 10 days only. On the third leaf the fungus rarely formed stromata and never sporulated. The fourth leaf already showed complete ontogenetic resistance. In the diploid variety Priscilla the gene Vf, which confers resistance against scab to apple leaves, was expressed as reduced stroma formation without sporulation, as in the case of ontogenetic resistance. In the triploid variety Sir Prize with the gene Vf, stroma formation was retarded and colonization and sporulation occurred later on the first leaf, similarly to the reaction of the second leaf of variety Golden Delicious.     

Use of green fluorescent protein to quantify the growth of Colletotrichum during infection of tobacco

To develop a quantitative assay of fungal growth inside plant tissues, strains of Colletotrichum destructivum and Colletotrichum orbiculare were transformed with a modified green fluorescent protein (GFP) gene fused with a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus nidulans. Transformants expressed GFP in culture and had the same growth rate and general appearance as the wild type. GFP was observed in all fungal structures during infection of leaves of Nicotiana benthamiana, except for the melanized appressoria and setae. The timing and appearance of the fungal structures in the host appeared to be identical to that of the wild type. GFP accumulation in inoculated leaves of N. benthamiana was quantified in leaf extracts using a fluorescence microplate reader, and the quantity of fluorescence was strongly correlated with the growth of the fungus as measured by the amount of fungal actin gene expression using Northern blot hybridizations. These results demonstrated that assaying green fluorescence levels from a GFP-transformed fungus is an accurate, fast and easy means of quantifying fungal growth inside host plant cells.     

Genetic analysis of a mutation on appressorium formation in Magnaporthe grisea

The rice blast fungus, Magnaporthe grisea, forms a dome-shaped and darkly pigmented infection structure, an appressorium, to penetrate its host. Differentiation and maturation of appressoria are critical steps for successful infection. A spontaneous developmental mutant (MG01) defective in appressorium formation was found in this fungus. The mutant did not form appressoria either on inductive hydrophobic surfaces or on rice leaves. The addition of cyclic AMP or 1,16-hexadecanediol was not effective in inducing appressorium formation in this mutant. This mutant did not cause lesions on rice when inoculated with conidial suspension by spraying or injecting into the leaf sheath. Genetic analysis of the mutant indicated that the phenotype is under single gene control, designated APP5. Crosses with previously described appressorium defective mutants (app1⁻ and app3⁻) of Magnaporthe grisea suggested that the mutations are at different loci. Bulked segregant analysis was employed to obtain DNA markers linked to the APP5 locus.     

Microscopy and proteomic analysis of the non-host resistance of <Emphasis Type="Italic">Oryza sativa</Emphasis> to the wheat leaf rust fungus, <Emphasis Type="Italic">Puccinia triticina</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis>

Rice (Oryza sativa) cv. Nipponbare expresses non-host resistance (NHR) to the wheat leaf rust fungus, Puccinia triticina f. sp. tritici (Ptt). When the leaves of cv. Nipponbare were inoculated with Ptt, approx 93% of the urediniospores germinated on the leaf surface, but only 10% of the germinated spores formed appressoria over the stomata at one day post inoculation (1 dpi). Hydrogen peroxide (H₂O₂) accumulated in host cells around the appressoria at 3 dpi. Approx. 3% of the appressoria produced short hyphae inside the leaf, and fluorescence was observed in tissue invaded by the hyphae by 7 dpi. At 22 dpi, 0.2% of the sites with appressoria formed branching infection hypha in mesophyll cells, but no substomatal vesicles, haustorial mother cells or haustoria were observed. Proteins were extracted from leaves 3 dpi and analyzed by two-dimensional gel electrophoresis (2-DE). A total 33 spots were reproducibly up-regulated and 9 were down-regulated by infection compared to the water inoculated control. Of these, 30 were identified by MALDI-TOF Mass Spectrometry. The identified proteins participate in defense/stress responses, energy/carbohydrate metabolism, oxidation–reduction processes, protein folding/turnover/cleavage/degradation, signal transduction and cell death regulation. The results indicates that NHR of rice to Ptt is consistent with a shift in protein and energy metabolism, increased antimicrobial activities, possibly including phytoalexin accumulation and cell wall reinforcement, increased cell repair, antioxidive and detoxification reactions, and enhanced prevention of plant cell death. Nearly half of the up-regulated identified proteins were associated with chloroplast and mitochondrial physiology suggesting important roles for these organelles during NHR.     

A method for staining infection hyphae in pine leaves.

Fungus-inoculated Pinus radiata leaves were fixed and then stained with periodic acid-Schiff reagent. Pieces of leaf with fungal material on the surface were removed. These pieces were stained in lactophenol cotton blue for a few minutes and then mounted in dilute lactophenol cotton blue. Microscopic examination of fungal material inside and outside the mounted leaf pieces revealed the following: condidia and germ tubes on the leaf surface were red, appressoria remained unstained, and infection hyphae within the leaf were stained blue. This differential staining method was particularly useful for distinguishing germ tubes from infection hyphae arising from appressoria.     

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.     

A Method for Staining Infection Hyphae in Pine Leaves

Fungus-inoculated Pinus radiata leaves were fixed and then stained with periodic acid-Schiff reagent. Pieces of leaf with fungal material on the surface were removed. These pieces were stained in lactophenol cotton blue for a few minutes and then mounted in dilute lactophenol cotton blue. Microscopic examination of fungal material inside and outside the mounted leaf pieces revealed the following: conidia and germ tubes on the leaf surface were red, appressoria remained unstained, and infection hyphae within the leaf were stained blue. This differential staining method was particularly useful for distinguishing germ tubes from infection hyphae arising from appressoria.     

Pathogenesis of barley leaves by Helminthosporium teres II: Carbohydrate involvement

Conidia of Helminthosporium teres had negligible difference in germination and germ tube length between the decolorized and non-decolorized host leaves. Appressoria, penetration and colonization were less on decolorized host leaves, but addition of exogenous nutrients stimulated these stages. Leached conidia had reduced germination on decolorized host leaves, while appressoria formation, penetration and colonization were negligible. The addition of nutrients in the external environment, however, enabled some of the leached conidia to penetrate and colonize. Stimulation by the exogenous nutrients in decreasing order were: sucrose > glucose > yeast extract > leaf leachates. Optimum levels for various nutrients tested were 2% (w/v) each of sucrose and glucose, and 0.1% (w/v) yeast extract. Higher concentrations inhibited these stages of infection. Leached conidia and decolorized leaves had smaller amounts of carbohydrates than non-leached conidia and non-decolorized leaves, respectively. Depletion of host carbohydrates reduced appressoria formation, penetration and colonization and loss of carbohydrates from spores reduced germination.     

Abnormal germling development by brown rust and powdery mildew on cer barley mutants

The barley leaf rust fungus forms appressoria over host leaf stomata and penetrates via the stomatal pore. High levels of avoidance to leaf rust fungi have been described in some wild accessions of Hordeum species where a prominent wax layer on the stomata inhibits triggering of fungal appressorium differentiation. Leaf rust avoidance has not yet been found in H. vulgare. Since cuticular leaf waxes are implicated in the avoidance trait, we screened 27 eceriferum (cer) mutant lines of H. vulgare for avoidance to barley leaf rust. These mutations affect leaf waxes. Reduction in numbers of germ tubes forming appressoria over stomata was found in some lines, but the greatest reduction (ca 30%) was less than previously found in wild barley spp. or in an accession of H. chilense used here as a check. In one line (cer-zh654), avoidance was due to a combination of factors. Firstly, fewer germ tubes oriented towards stomata and so failed to contact them. Secondly, some germ tubes that encountered stomata did not form appressoria but over-grew them. In this line, therefore, the fungus tended to fail both to locate and to respond to stomata. The appressoria of barley powdery mildew form on leaf epidermal cells that they penetrate directly. On certain cer lines, a proportion of germlings of the barley powdery mildew fungus developed abnormally, suggesting that germlings failed to recognise and/or respond to the leaf surface waxes on these mutants.