Subcuticular Hyphae Research Articles

A Melanin-Deficient Isolate of Venturia inaequalis Reveals Various Roles of Melanin in Pathogen Life Cycle and Fitness.

Venturia inaequalis is the ascomycetous pathogen causing apple scabs and forms dark-pigmented spores and partially melanised infection structures. Although melanin is considered to be essential for the infection of host tissue, a spontaneously occurring melanin-deficient mutant was isolated from an abaxial side of an apple leaf and can be cultivated in vitro as well as in vivo. The morphology and development of the melanin-deficient-isolate SW01 on leaves of susceptible apple plants were compared to that of the corresponding wild-type isolate HS1. White conidia of SW01 were often wrinkled when dry and significantly increased their volume in suspension. Germination and formation of germtubes and appressoria were not impaired; however, the lack of melanisation of the appressorial ring structure at the interface with the plant cuticle significantly reduced the infection success of SW01. The colonisation of leaf tissue by non-melanised subcuticular hyphae was not affected until the initiation of conidiogenesis. Non-melanised conidiophores penetrated the plant cuticle from inside less successfully than the wild type, and the release of white conidia from less solid conidiophores above the cuticle was less frequent. Melanin in the outer cell wall of V. inaequalis was not required for the survival of conidia under ambient temperature or at -20 °C storage conditions, however, promoted the tolerance of the pathogen to copper and synthetic fungicides affecting the stability and function of the fungal cell wall, plasma membrane, respiration (QoIs) and enzyme secretion, but had no effect on the sensitivity to sulphur and SDHIs. The roles of melanin in different steps of the V. inaequalis life cycle and the epidemiology of apple scabs are discussed.

Cytological Observation of the Infectious Process of Venturia carpophila on Peach Leaves

Peach scab caused by Venturia carpophila is one of the most destructive fungal diseases of peach worldwide, and it seriously affects peach production. Until now,the infectious process and pathogenesis of V. carpophila on peach have remained unclear. Here we present the infection behavior of V. carpophila at the ultrastructural and cytological levels in peach leaves with combined microscopic investigations (i.e., light microscopy, confocal laser scanning microscopy, scanning electron microscopy, and transmission electron microscopy). V. carpophila germinated at the tip of conidia and produced short germ tubes on peach leaf surfaces at 2 days post inoculation (dpi). At 3 dpi, swollen tips of germ tubes differentiated into appressoria. At 5 dpi, penetration pegs produced by appressoria broke through the cuticle layer and then differentiated into thick subcuticular hyphae in the pectin layer of the epidermal cell walls. At 10 dpi, the subcuticular hyphae extensively colonized in the pectin layer. The primary hyphae ramified into secondary hyphae and proliferated along with the incubation. At 15 dpi, the subcuticular hyphae divided laterally to form stromata between the cuticle layer and the cellulose layer of the epidermal cells. At 30 dpi, conidiophores developed from the subcuticular stromata. Finally, abundant conidiophores and new conidia appeared on leaf surfaces at 40 dpi. These results provide useful information for further a understanding of V. carpophila pathogenesis.

Induced expression of the Fragaria × ananassa Rapid alkalinization factor-33-like gene decreases anthracnose ontogenic resistance of unripe strawberry fruit stages.

SummaryRapid alkalinization factor (RALF) genes encode for ubiquitous small peptides that stimulate apoplastic alkalinization through interaction with malectin‐like receptor kinase. RALF peptides may act as negative regulators of plant immune response, inhibiting the formation of the signal receptor complex for immune activation. Recently RALF homologues were identified in different fungal pathogen genomes contributing to host infection ability. Here, FaRALF‐33‐like gene expression was evaluated in strawberry fruits inoculated with Colletotrichum acutatum, Botrytis cinerea, or Penicillium expansum after 24 and 48 h post‐infection. To investigate the role of FaRALF‐33‐like in strawberry susceptibility, transient transformation was used to overexpress it in white unripe fruits and silence it in red ripe fruits. Agroinfiltrated fruits were inoculated with C. acutatum and expression, and histological analysis of infection were performed. Silencing of FaRALF‐33‐like expression in C. acutatum‐inoculated red fruits led to a delay in fruit colonization by the fungal pathogen, and infected tissues showed less penetrated infective hyphae than in wild‐type fruits. In contrast, C. acutatum‐inoculated white unripe fruits overexpressing the FaRALF‐33‐like gene decreased the ontogenic resistance of these fruits, leading to the appearance of disease symptoms and penetrated subcuticular hyphae, normally absent in white unripe fruits. The different response of transfected strawberry fruits to C. acutatum supports the hypothesis that the FaRALF‐33‐like gene plays an important role in the susceptibility of fruits to the fungal pathogen C. acutatum.

Mechanisms of Broad Host Range Necrotrophic Pathogenesis in Sclerotinia sclerotiorum.

Among necrotrophic fungi, Sclerotinia sclerotiorum is remarkable for its extremely broad host range and for its aggressive host tissue colonization. With full genome sequencing, transcriptomic analyses and the increasing pace of functional gene characterization, the factors underlying the basis of this broad host range necrotrophic pathogenesis are now being elucidated at a greater pace. Among these, genes have been characterized that are required for infection via compound appressoria in addition to genes associated with colonization that regulate oxalic acid (OA) production and OA catabolism. Moreover, virulence-related secretory proteins have been identified, among which are candidates for manipulating host activities apoplastically and cytoplasmically. Coupled with these mechanistic studies, cytological observations of the colonization process have blurred the heretofore clear-cut biotroph versus necrotroph boundary. In this review, we reexamine the cytology of S. sclerotiorum infection and put more recent molecular and genomic data into the context of this cytology. We propose a two-phase infection model in which the pathogen first evades, counteracts and subverts host basal defense reactions prior to killing and degrading host cells. Spatially, the pathogen may achieve this via the production of compatibility factors/effectors in compound appressoria, bulbous subcuticular hyphae, and primary invasive hyphae. By examining the nuances of this interaction, we hope to illuminate new classes of factors as targets to improve our understanding of broad host range necrotrophic pathogens and provide the basis for understanding corresponding host resistance.

Oxaloacetate acetylhydrolase gene mutants of Sclerotinia sclerotiorum do not accumulate oxalic acid, but do produce limited lesions on host plants.

The oxaloacetate acetylhydrolase (OAH, EC 3.7.1.1)-encoding gene Ss-oah1 was cloned and functionally characterized from Sclerotinia sclerotiorum. Ss-oah1 transcript accumulation mirrored oxalic acid (OA) accumulation with neutral pH induction dependent on the pH-responsive transcriptional regulator Ss-Pac1. Unlike previously characterized ultraviolet (UV)-induced oxalate-deficient mutants ('A' mutants) which retain the capacity to accumulate OA, gene deletion Δss-oah1 mutants did not accumulate OA in culture or during plant infection. This defect in OA accumulation was fully restored on reintroduction of the wild-type (WT) Ss-oah1 gene. The Δss-oah1 mutants were also deficient in compound appressorium and sclerotium development and exhibited a severe radial growth defect on medium buffered at neutral pH. On a variety of plant hosts, the Δss-oah1 mutants established very restricted lesions in which the infectious hyphae gradually lost viability. Cytological comparisons of WT and Δss-oah1 infections revealed low and no OA accumulation, respectively, in subcuticular hyphae. Both WT and mutant hyphae exhibited a transient association with viable host epidermal cells at the infection front. In summary, our experimental data establish a critical requirement for OAH activity in S. sclerotiorum OA biogenesis and pathogenesis, but also suggest that factors independent of OA contribute to the establishment of primary lesions.

The mannose‐binding lectin gene FaMBL1 is involved in the resistance of unripe strawberry fruits to Colletotrichum acutatum

The fungal pathogen Colletotrichum acutatum is the causal agent of strawberry (Fragaria × ananassa) anthracnose. Although the fungus can infect strawberry fruits at both unripe and ripe stages, the symptoms appear only on red ripe fruits. On white unripe fruits, the pathogen becomes quiescent as melanized appressoria after 24 h of interaction. Previous transcriptome analysis has indicated that a mannose-binding lectin (MBL) gene is the most up-regulated gene in 24-h-infected white strawberries, suggesting a role for this gene in the low susceptibility of unripe stages. A time course analysis of the expression of this MBL gene, named FaMBL1 (Fragaria × ananassa MBL 1a), was undertaken to monitor its expression profile in white and red fruits at early interaction times: FaMBL1 was expressed exclusively in white fruit after 24 h, when the pathogen was quiescent. Agrobacterium-mediated transient transformation was used to silence and overexpress the FaMBL1 gene in 24-h-infected white and red strawberries, respectively. FaMBL1-silenced unripe fruits showed an increase in susceptibility to C. acutatum. These 24-h-infected tissues contained subcuticular hyphae, indicating pathogen penetration and active growth. In contrast, overexpression of FaMBL1 in ripe fruits decreased susceptibility; here, 24-h-infected tissues showed a high percentage of ungerminated appressoria, suggesting that the growth of the pathogen had slowed. These data suggest that FaMBL1 plays a crucial role in the resistance of unripe strawberry fruits to C. acutatum.

Penetration Behaviour of Venturia nashicola, Associated with Hydrogen Peroxide Generation, in Asian and European Pear Leaves

AbstractThe penetration behaviour of the pathogen Venturia nashicola, which causes scab disease in Asian pears, was studied at the ultrastructural and cytochemical levels in host and non‐host leaves. We show, for the first time, that before V. nashicola penetrated the cuticle of the epidermis of the pear leaf, the appressorial bottom of the pathogen invaginated to form a cavity that contains electron‐dense material. The leaf cuticle beneath the cavity also became highly electron dense following penetration by V. nashicola. The location of these electron‐dense materials at the sites of penetration of the pathogen into plant cell walls suggests that they might be related to enzymes capable of degrading cell walls and that the cavities might be needed for successful penetration of leaves by V. nashicola. The generation of hydrogen peroxide (H2O2) was observed in penetration‐related infection structures of V. nashicola, such as appressorial bottoms, infection sacs, penetration pegs and necks of subcuticular hyphae regardless of whether the interaction of V. nashicola with pear plants was compatible or incompatible. Nonetheless, more H2O2 was generated at the sites of the structures in scab‐inoculated susceptible leaves than that in scab‐inoculated resistant ones. Furthermore, the decrease in the level of H2O2 generation following treatment with the antioxidant ascorbic acid partially prevented the penetration of the cuticle. Therefore, the generation of H2O2 from the penetration‐related structures might be a pathogenicity factor that contributes to strengthening the penetration peg of V. nashicola.

Oxalic Acid Has an Additional, Detoxifying Function in Sclerotinia sclerotiorum Pathogenesis

The mechanism of the diseases caused by the necrotroph plant pathogen Sclerotinia sclerotiorum is not well understood. To investigate the role of oxalic acid during infection high resolution, light-, scanning-, transmission electron microscopy and various histochemical staining methods were used. Our inoculation method allowed us to follow degradation of host plant tissue around single hyphae and to observe the reaction of host cells in direct contact with single invading hyphae. After penetration the outer epidermal cell wall matrix appeared degraded around subcuticular hyphae (12-24 hpi). Calcium oxalate crystals were detected in advanced (36-48 hpi) and late (72 hpi) infection stages, but not in early stages. In early infection stages, surprisingly, no toxic effect of oxalic acid eventually secreted by S. sclerotiorum was observed. As oxalic acid is a common metabolite in plants, we propose that attacked host cells are able to metabolize oxalic acid in the early infection stage and translocate it to their vacuoles where it is stored as calcium oxalate. The effects, observed on healthy tissue upon external application of oxalic acid to non-infected, living tissue and cell wall degradation of dead host cells starting at the inner side of the walls support this idea. The results indicate that oxalic acid concentrations in the early stage of infection stay below the toxic level. In plant and fungi oxalic acid/calcium oxalate plays an important role in calcium regulation. Oxalic acid likely could quench calcium ions released during cell wall breakdown to protect growing hyphae from toxic calcium concentrations in the infection area. As calcium antimonate-precipitates were found in vesicles of young hyphae, we propose that calcium is translocated to the older parts of hyphae and detoxified by building non-toxic, stable oxalate crystals. We propose an infection model where oxalic acid plays a detoxifying role in late infection stages.

Evaluation of Response to Pecan Scab Pathogen Race Using a Detached Leaflet Protocol

A detached leaflet protocol was developed for the evaluation of resistance to Fusicladium effusum in a seedling pecan population segregating for resistance. Leaflets at half to full expansion were detached from seedling trees, sprayed with a conidial suspension (isolate De-Tif-3), placed in a polyethylene bag in a growth chamber, and evaluated microscopically 7 and 14 days after inoculation. The percentage germinated conidia producing subcuticular hyphae was the best determinant of susceptibility with genotypes producing more than 15% subcuticular hyphae considered susceptible. Leaflets at half expansion had higher percentages of subcuticular hyphae and gave a clearer separation between susceptible and resistant genotypes than leaflets at full expansion. An evaluation period of 14 days was preferable to 7 days to allow slower reacting genotypes to be better evaluated. The detached leaflet protocol was evaluated in contrasting environments and was found to be robust to differences in shading and leaflet wetness. Detached leaflet tests gave similar results to field inoculations but were superior in consistently detecting susceptible genotypes. This protocol will be useful in evaluating the inheritance of pecan leaf scab resistance in breeding progenies.

An unusual method of development and sporulation of Colletotrichum gloeosporioides on castor leaf – an SEM account

Development and sporogenesis of Colletotrichum gloeosporioides on castor leaf differed from that on other known host plants. C. gloeosporioides had three kinds of hyphae on castor leaf: primary infection hyphae (PIH), runner hyphae (RH) and secondary infection hyphae (SIH). The PIH originated from conidia, grew on leaf surface and entered the leaf by direct penetration of the cuticle without forming appressoria. The RH were sub-cuticular hyphae, the track of which was traceable by the bulgings on the leaf surface, and the SIH were the hyphae that emerged to leaf surface from RH through the cuticle or stomata. Conidia were initiated as small protrusions along the lengths of RH and SIH that got differentiated into distinct conidia, each born on a short stumpy conidiophore without forming any congregation. The protrusions from RH emerged to the leaf surface by piercing the cuticle, and they developed into distinct conidia on the leaf surface. The conidia developed from RH and SIH were identical in size and shape. Even though conidia were occasionally found emerged through stomata, that appeared to be random than a preferred route for the discharge of conidia. The penetration and sporogenesis of C. gloeosporioides on castor leaf differed from that reported on mulberry leaf.

Inhibitory Effects of Antioxidant Reagent in Reactive Oxygen Species Generation and Penetration of Appressoria of Alternaria alternata Japanese Pear Pathotype

In the Japanese pear pathotype of Alternaria alternata, H2O2 is generated solely from penetration pegs and not from other portions of subcuticular hyphae within the pectin layers of host leaves. A close association between H2O2 generation and fungal aggressiveness is expected because the pegs are important for fungal penetration into the host epidermis. To determine the potential role of reactive oxygen species in microbial pathogenicity, we studied the inhibitory effects of the antioxidant reagent ascorbic acid and the NADPH oxidase inhibitor diphenylene iodonium on infection of the pathogen. In our study, we showed H2O2 generation to be inhibited by inoculation with the mixture of ascorbic acid or diphenylene iodonium and spores at the pegs in the spore-inoculated host leaves. The decrease of generation in the pegs resulted in penetration failure, indicating that H2O2 generation probably contributed to strengthening of the penetration and probably was recruited by NADPH oxidase.

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.

An in vitro method for the analysis of infection‐related morphogenesis in Fusarium graminearum

Fusarium graminearum is a significant pathogen of many cereal crops. With its genetic tractability, ease of culture, genome sequence availability and economic significance, F. graminearum has become the subject of intensive molecular research. Although molecular tools have been developed to enhance research into virulence determinants of F. graminearum, simple assays for infection-related development are lacking. As such, the objective of this study was to develop an in vitro protocol for the analysis of infection-related morphogenesis in F. graminearum. We demonstrate that two morphologically distinct hyphal structures are produced by F. graminearum during the invasion of detached wheat glumes: subcuticular hyphae and bulbous infection hyphae. Specialized wheat epidermal cells (papillae) appear to act as sites of invasion by F. graminearum on the adaxial side of detached wheat glumes. In addition, the development of bulbous infection hyphae is dependent on the pathogenicity mitogen-activated protein kinase Gpmk1, further supporting the infection-related nature of these structures. This relatively simple assay will contribute to the tractability of the F. graminearum system and help to uncover molecular requirements for infection-related development.

Infection process of Colletotrichum dematium on mulberry leaves: An unusual method of sporulation

The pre-penetration and infection process of Colletotrichum dematium on mulberry leaf was investigated by scanning electron microscope. Conidia produced on germination appressoria directly or at the end of short germ tubes. Appressoria were formed mostly over cuticle, but sometimes over stomata also. At 72 h post-inoculation, an extensive network of sub-cuticular runner hyphae (RH) was produced. The RH were traceable by the cuticular bulgings on leaf surface. The RH emerged to leaf surface through ruptured cuticle to form secondary infection hyphae (SIH). The SIH re-entered the leaf tissue by sending penetration branches through stomata. Conidia were formed singly on short conidiophores from the RH and SIH, at short intervals. The conidia developed on RH were exposed to leaf surface through ruptured cuticle. Some times conidia were released through stomata also. The RH and SIH had thick knots from which hyphal branches and conidia were developed. Definite acervuli were not developed.

Ultrastructural Study on Acibenzolar-S-Methyl-Induced Scab Resistance in Epidermal Pectin Layers of Japanese Pear Leaves

The infection behavior of Japanese pear scab pathogen Venturia nashicola race 1 was studied ultrastructurally in acibenzolar-S-methyl (ASM)-pretreated susceptible Japanese pear (cv. Kousui) leaves to determine the mechanism of ASM-induced scab resistance. On ASM-pretreated leaf surfaces, the infection behavior (conidial germination and appressorial formation) was similar to that on distilled water (DW)-pretreated leaves prior to cuticle penetration by the pathogen. However, after penetration, differentiated behavior was found in epidermal pectin layers and middle lamellae of the ASM-pretreated leaves. Subcuticular hyphae in epidermal pectin layers and middle lamellae of ASM-pretreated pear leaves were observed at lower frequency than in DW-treated leaves. The results indicated that fungal growth was suppressed in ASM-pretreated pear leaves. In the pectin layers of ASM- and DW-pretreated leaves, some hyphae showed morphological modifications, which were used as criteria to judge collapse of hyphal cells, including plasmolysis, necrotic cytoplasm, and cell wall destruction. More hyphae had collapsed in ASM-pretreated leaves than in DW-treated ones. In addition, the cell walls of collapsed hyphae broke into numerous fibrous and amorphous pieces, suggesting that ASM-induced scab resistance might be associated with cell-wall-degrading enzymes from pear plants. In addition, results from morphometrical analysis suggested that the activity or production of pectin-degrading enzyme from hyphae were inhibited by ASM application when compared with DW treatment.

Ultrastructural study on scab resistance expressed in epidermal pectin layers of pear leaves

Proliferation and collapse of subcuticular hyphae of Venturia nashicola race 1 were studied ultrastructurally, after inoculation of susceptible Japanese pear cv. Kousui, resistant Japanese pear cv. Kinchaku, resistant Asian pear strain Mamenashi 12 and nonhost European pear cv. Flemish Beauty leaves, to understand the nature of the resistance mechanism. After cuticle penetration by the pathogen, the hyphae were observed at lower frequency in epidermal pectin layers and middle lamellae of leaves of the three resistant plants than in those of susceptible ones. This result suggested that fungal growth was suppressed in the incompatible interaction between pear and V. nashicola race 1. In the pectin layers of all inoculated plants, some hyphae had modifications such as breaks in the plasmalemma with plasmolysis, necrotic cytoplasm and degraded cell walls. More hyphae had collapsed in the leaves of the three resistant plants than in those of the susceptible cv. Kousui. In collapsed hyphae, the polymerized cell walls broke into numerous fibrous and amorphous pieces, showing that the scab resistance might be associated with cell wall-degrading enzymes from pear plants.

Heteroconium and Pirozynskiella n. gen., with comments on conidium transseptation

Heteroconium citharexyli, the type species of this genus, is illustrated and redescribed as a sooty mold bearing acropetal chains of conidia showing a basifugal sequence of septation. Heteroconium neriifoliae, H. glutinosa and the Heteroconium synanamorph of Antennulariella concinna are congeneric. The latter species is neotypified, illustrated and described. Pirozynskiella new genus, typified by P. solaninum comb. nov. (≡ Helminthosporium solaninum), differs from Heteroconium in having an obligate association with asterinaceous fungi and in the centrifugal sequence of conidium transseptation after the initial median septum. Pirozynskiella costaricensis comb. nov. (≡ Dendryphion costaricensis) is illustrated and described. Heteroconium tetracoilum and H. chaetospira are fungicoles of wood and bark; the former has basifugal conidium septation and the latter has a centrifugal sequence. The two latter species can be excluded from the Heteroconium. Basifugal and centrifugal septation of conidia is discussed with reference to several sooty molds, to some foliicolous anamorphs with subcuticular hyphae (Heterosporiopsis) and to some wood and bark hyphomycetes (Septonema, Taeniolella). Ten other species included in Heteroconium are known to me only from their original descriptions; only one (H. asiaticum) is probably a sooty mold.

Heteroconium and Pirozynskiella n. gen., with comments on conidium transseptation

Heteroconium citharexyli, the type species of this genus, is illustrated and redescribed as a sooty mold bearing acropetal chains of conidia showing a basifugal sequence of septation. Heteroconium neriifoliae, H. glutinosa and the Heteroconium synanamorph of Antennulariella concinna are congeneric. The latter species is neotypified, illustrated and described. Pirozynskiella new genus, typified by P. solaninum comb. nov. (-Helminthosporium solaninum), differs from Heteroconium in having an obligate association with asterinaceous fungi and in the centrifugal sequence of conidium transseptation after the initial median septum. Pirozynskiella costaricensis comb. nov. (-Dendryphion costaricensis) is illustrated and described. Heteroconium tetracoilum and H. chaetospira are fungicoles of wood and bark; the former has basifugal conidium septation and the latter has a centrifugal sequence. The two latter species can be excluded from the Heteroconium. Basifugal and centrifugal septation of conidia is discussed with reference to several sooty molds, to some foliicolous anamorphs with subcuticular hyphae (Heterosporiopsis) and to some wood and bark hyphomycetes (Septonema, Taeniolella). Ten other species included in Heteroconium are known to me only from their original descriptions; only one (H. asiaticum) is probably a sooty mold.

Pathogenic Variation of Cladosporium caryigenum Isolates and Corresponding Differential Resistance in Pecan

To facilitate the breeding of scab-resistant pecan [Carya illinoinensis (Wangenh.) C. Koch] cultivars, more information is needed about the pathogenic variation of the causal organism, Cladosporium caryigenum (Ell. et Lang.) Gottwald (1982). This study examined the virulence patterns resulting from the field inoculations of 19 pecan cultivars with 12 monoconidial isolates from 8 pecan cultivars at 7 locations. The virulence pattern was different for each of the fungal isolates. Each isolate was virulent on some cultivars and avirulent on others. Most isolates were most virulent on the cultivar of their origin and one or a few other cultivars. Several cultivars were resistant to most of the tested isolates. The results indicate a large pool of differential and ephemeral resistance to scab resides in the pecan germplasm. Microscopic examination of developing lesions suggests that resistance is mediated by the speed of plant cell wall modifications that limit the growth of subcuticular hyphae. This information will be useful in the selection of cultivars with functionally different resistance genes for use in designing cultivar mixtures or in breeding programs.

A Detached Leaf Technique for Studying Race-specific Resistance to Cladosporium caryigenum in Pecan

A detached leaf screening technique was developed for studying specific interactions between pecan [Carya illinoinensis (Wangenh.) C. Koch] cultivars and isolates of the pecan scab fungus, Cladosporium caryigenum. Monoconidial isolates were obtained from leaf scab lesions on `Wichita', `Desirable', `Cape Fear', and `Elliot'. Each isolate was then inoculated onto detached leaves of each of the four cultivars and fungal growth was observed under the microscope after eight days. `Wichita', `Desirable', and `Cape Fear' isolates produced subcuticular hyphae at a much higher frequency when inoculated back onto the cultivar from which they were isolated in comparison to the other cultivars. The `Elliot' isolate was able to produce a high frequency of subcuticular hyphae when inoculated onto `Elliot' and `Cape Fear', but not when inoculated onto `Desirable' and `Wichita'. Field inoculations conducted with the `Wichita' and `Desirable' isolates validated the detached leaf protocol. The results obtained indicate that pecan scab is composed of multiple races with a high degree of specificity for host cultivars. A rapid whole-leaf staining system is presented which appears to have wide applicability to assessing fungal growth in leaves.