The Course of Colonization of Two Different Vitis Genotypes by Plasmopara viticola Indicates Compatible and Incompatible Host-Pathogen Interactions

ABSTRACT The course of colonization of leaf mesophyll by the causal agent of grapevine downy mildew, Plasmopara viticola, in a susceptible and a resistant grapevine genotype was examined in order to characterize the development of the pathogen in compatible and incompatible host-pathogen interactions. Within a few hours after inoculation, the pathogen was established in the susceptible Vitis vinifera cv. Müller-Thurgau and formed primary hyphae with a first haustorium. No further development occurred in the following 10 to 18 h. The next step, in which the hyphae grew and branched to colonize the intercellular space of the host tissue, was observed 1.5 days after inoculation. After 3 days, the intercostal fields were entirely filled with mycelium and sporulation was abundant under favorable environmental conditions. The first infection steps were essentially the same in the resistant V. rupestris. However, the invasive growth of P. viticola was delayed, and further development ceased before the intercostal fields were filled with mycelium.     

Primary Haustorial Development of Striga asiatica on Host and Nonhost Species

ABSTRACT Initial interactions of Striga asiatica with a susceptible host and non-host plants were examined by histological methods. Haustorial development was initiated when radicles of S. asiatica were placed in contact with host or nonhost roots. Reorganization of the S. asiatica root apical meristem was rapid and involved the formation of a distal group of cells that penetrated the host or nonhost root. Penetration of the epidermis of the host (sorghum) roots and advance into the cortex occurred within 24 to 48 h of inoculation. Penetration of the endodermis by the developing endophyte was delayed for 72 to 96 h after initial contact. However, upon penetration vascular continuity was established between parasite and host. In contrast, interactions with nonhosts provided evidence of active resistance mechanisms. Penetration of lettuce, marigold, and cowpea roots by S. asiatica was most frequently arrested in the cortex, and endophytic cells were necrotic 72 h after inoculation. Some species-specific differences were observed in the reactions of nonhosts to penetration, although in their general nature the interactions with S. asiatica were similar.     

鲜食葡萄品种对霜霉病的抗性及抗病机理研究

采用田间自然发病调查，田间接种鉴定和离体葡萄叶圆片室内接种鉴定鲜食葡萄品种对霜霉病的抗性及其抗性机理研究表明，品种间对霜霉病的抗性存在显著差异。可依感病病级分为三类，低感品种有“z工伊豆”、“奥林匹亚”、“山东早红”、“森田尼”、“早玛瑙”。几乎所有的无核品种均高感霜霉病，葡萄对霜霉病的抗性与叶内可滴定酸、还原糖、游离氨基酸含量无显著相关，与叶背气也密度、气孔开度也无相关，而外施高浓度邻苯二酚却显著降低了霜霉病发病程度。     

Hypersensitive cell death and post-haustorial defence responses arrest the orange rust (Hemileia vastatrix) growth in resistant coffee leaves

The growth of a coffee orange rust fungus (Hemileia vastatrix Berk and Br.) isolate (race II) and the sequence of responses it induced in leaves of resistant Coffea arabica L. and C. congensis Froehner as well as on a susceptible C. arabica were investigated cytologically and biochemically. The percentages of germinated urediospores and of appressoria formed over stomata as well as the fungal growth inside leaf tissues were similar in resistant and susceptible leaves until the 3rd day after the inoculation. In the susceptible leaves, at the majority of the infection sites (70%) the fungus pursued its growth without apparent inhibition while in the resistant leaves the fungus ceased its growth with higher frequency (34% in C. arabica and 54% in C. congensis) after the formation of at least one haustorium. The first signs of incompatibility, detected 2 days after the inoculation, were cytologically expressed by hypersensitive host cell death (HR), host cell wall autofluorescence and haustoria encasement with callose and β-1,4-glucans. Biochemically, two peaks of phenylalanine ammonia-lyase (PAL) activity were detected by 2 and 5 days after the inoculation. The 1st peak coincided with the early accumulation of phenolic compounds and with the beginning of cell death. The 2nd peak could be related to later accumulation of phenols and the lignification of the host cell walls. About 5–7 days after the inoculation, ultrastructural observations revealed the accumulation of a material partially crystallized in the intercellular spaces around the senescent hyphae, next to dead host cells and in close association with the middle lamella that initially labelled for pectins. It also contained polysaccharides and phenolic-like compounds. Cellulose, hemicellulose, extensins, hydroxyproline-rich glycoproteins and proteins were not detected. The hypertrophy of the host cells in the infection area were also observed around 12 days after the inoculation corresponding macroscopically to the reaction flt.In susceptible plants, cell death was also observed 3 days after the inoculation but only in a reduced percentage of infection sites in which the fungus aborted at an early stage. A late haustorium encasement and stimulation of PAL activity were also observed but these delayed host responses did not prevent fungal growth and sporulation.The intercellular material, only observed in the resistant plants, is here reported for the first time and although its role is unknown it might be the result of plant cell death.     

A standardised method for the quantitative analysis of resistance to grapevine powdery mildew

Powdery mildew caused by Erysiphe necator is one of the most important diseases affecting grapevine (Vitis vinifera, L.). Control of this pathogen is based on the use of fungicides, which cause environmental damage and increase production costs. A cost-effective and environmentally friendly alternative to control the disease relies on using resistant varieties. While most V. vinifera cultivars are susceptible to powdery mildew, several species belonging to the Vitaceae have been described as resistant. Several loci for resistance to grapevine powdery mildew have been identified through genetic analysis of segregating populations derived from different resistance sources. Identifying quantitative trait loci (QTL) with minor effects on the resistance may prove valuable in a strategy of pyramiding, which aims at increasing the durability of the resistance. However, current methods for evaluation of resistance either do not take into account quantitative variations, or, if they do, are not adapted to large sample sets. Here we develop a method for the analysis of quantitative resistance to grapevine powdery mildew in large populations. We devised a semi-quantitative resistance scale and confirmed the usefulness of a cell counter to quantify sporulation. We compared three inoculation methods and identified dry inoculation using a settling tower as the one giving the best infection. Finally, we confirmed the value of the method by applying it to a set of plants segregating for resistance to E. necator. Using the method described here for the quantitative analysis of the resistance to powdery mildew will prove valuable for breeding for durable resistance.     

Histopathology of compatibility and incompatibility between oilseed rape and Albugo Candida

Cotyledons of one resistant and three susceptible rape lines/cultivars were inoculated with zoospores of Albugo Candida race 7. Samples of whole cotyledons were examined by differential interference contrast microscopy. The time course of the infection process was followed histologically. Germination of zoospore cysts occurred 2-3 h after inoculation. Infection was initiated with germ-tubes penetrating through stomata. Haustorium formation was first observed in the palisade mesophyll cells adjacent to the substomatal chambers 8 h after inoculation.
Only after the establishment of the first haustorium did compatible and incompatible interactions begin to differentiate. In the resistant cultivar, most primary hyphae produced single haustoria. Necrosis of the invaded host cell was first observed 12 h after inoculation followed by cessation of fungal growth. The death of host cells was largely restricted to the penetration site; the adjacent non-penetrated cells remained apparently unaffected. In the susceptible hosts, necrosis of infected cells occurred only infrequently, and hyphal growth continued unabated, resulting in mycelial ramification into the mesophyll. Numerous haustoria were produced.
Histological studies showed that the earliest event distinguishing a compatible from an incompatible interaction occurred after formation of the first haustorium and that resistance was not manifested until the host mesophyll cell had come into contact with the first haustorium. The distinction between compatibility and incompatibility was substantiated by quantitative analysis of white rust development on both resistant and susceptible lines/cultivars.     

Histological responses of host and non-host plants to <Emphasis Type="Italic">Hyaloperonospora parasitica</Emphasis>

Differences in Hyaloperonospora parasitica development and plant tissue responses were compared for 10 cruciferous hosts (including both resistant and susceptible genotypes), 3 leguminous and 1 graminaceous non-host species. Cotyledons, or true leaves in the case of Triticum aestivum and Pisum sativum, were studied at 2, 8, 24 h and 3, 5, 7 days post inoculation (dpi). The high levels of zoosporangial germination observed on all species tested, as well as on glass slides, suggested that inhibition of germination did not play a significant role in distinguishing host versus non-host resistance. During the early stages of infection, at spore germination and host penetration, there was no evidence of a clear-cut difference between Brassica host species which displayed a hypersensitive, partially resistant or susceptible reaction compared with non-host species. Haustoria formation was the key infection phase for the establishment of biotrophy. Across all tested species, haustoria were initiated inside the epidermal cells. However, there were significant differences in the frequency and timing of haustorial formation and the final size of haustoria among the tested species at early infection stage. Fully developed haustoria were never observed in Raphanus raphanistrum, Triticum aestivum, Lupinus angustifolius nor Trifolium subterraneum. Instead, the haustorium development appears to abort in the penetrated epidermal cells of these species. Although haustoria were formed in the epidermal and mesophyll cells of Sinapsis alba and Pisum sativum, subsequent hyphal growth and/or continued haustoria formation were rare or few, respectively. Hypersensitive reaction was the key resistance response observed among the host and non-host resistant species tested. It is noteworthy that, in the initial stages of pathogenesis, there was no differentiating point that separated the non-host species from those that were hosts.     

Seasonal development of ontogenic resistance to downy mildew in grape berries and rachises

ABSTRACT Clusters of Vitis vinifera and V. labrusca are reported to become resistant to Plasmopara viticola at stages of development ranging from 1 to 6 weeks postbloom. It has been suggested that resistance is associated with loss of the infection court as stomata are converted to lenticels, but the time of onset, cultivar variation, and seasonal variation in ontogenic resistance has remained uncertain, as has the comparative susceptibility of stem tissue within the fruit cluster. In New York, we inoculated clusters of V. vinifera cvs. Chardonnay and Riesling and V. labrusca cvs. Concord and Niagara at stages from prebloom until 5 to 6 weeks postbloom. Berries were infected and supported profuse sporulation until 2 weeks postbloom, and pedicel tissue remained susceptible until 4 weeks postbloom. Although berries on later-inoculated clusters failed to support sporulation, discoloration and necrosis of berry tissues was often noted, and necrosis of the pedicel within such clusters often led to further discoloration, shriveling, reduced size, or loss of berries. When the epidermis of discolored berries that initially failed to support sporulation was cut, the pathogen emerged and sporulated through incisions, indicating that lack of sporulation on older symptomatic berries was due to infection at an early stage of berry development followed by conversion of functional stomata to lenticels during latency. We repeated the study on Chardonnay and Riesling vines in South Australia and found that the period of berry and rachis susceptibility was greatly increased. The protracted susceptibility of the host was related to the increased duration and phenological heterogeneity of bloom and berry development in the warmer climate of South Australia. The time of onset and subsequent expression of ontogenic resistance to P. viticola may thus be modified by climate and should be weighed in transposing results from one climatic area to another. Our results can be used to refine forecast models for grapevine downy mildew to account for changes in berry and rachis susceptibility, and to focus fungicide application schedules upon the most critical periods for protection of fruit.     

A microscopic study of the wheat-powdery mildew relationship after application of the systemic compounds procaine,griseofulvin and 6-azauracil

On wheat seedlings systemically protected by root application of procaine-hydrochloride, griseofulvin or 6-azauracil, germination of oidia ofErysiphe graminis f. sp.tritici and penetration of this fungus into the epidermal cell wall was as high as on control plants. Inhibition of powdery mildew development became apparent only after the penetration process had started. About 85% of the infections were halted within 24 hours after the inoculation, and did not result in the formation of a haustorium. In the other cases usually not more than one haustorium per infection court was formed, which often showed anomalies, characteristic for each compound used. Development of mycelium was scanty or absent and no sporulation occurred. Similarly, on plants of a resistant wheat variety, powdery mildew inhibition became apparent only after penetration of the host had started. There was no development of mycelium or sporulation. A severe reaction of certain epidermal cells to penetration by powdery mildew was observed on resistant as well as on treated and untreated susceptible plants. However, in relation to the total number of infections, the percentage of this type of reaction was low.     

Interaction between powdery mildew and barley with mlo5 mildew resistance

Powdery mildew infection of barley with the mlo5 barley powdery mildew resistance gene was examined, using near-isogenic barley lines, with and without mlo5 resistance, and two near-isogenic powdery mildew isolates, HL3/5 and GE3 with high (virulent) or low (avirulent) penetration efficiency on the resistant barley line. In all isolate–host combinations (except GE3 on the resistant barley line), frequency of haustorium formation increased significantly from zero at 11 h after inoculation to a maximum by 13 h, and there was no subsequent increase up to 24 h. In the susceptible barley line, 27% of appressoria from both isolates formed haustoria. Although this was significantly higher than the frequency of haustorium formation (18%) of HL3/5 on the resistant barley line, HL3/5 was much more successful than GE3 (frequency of haustorium formation less than 1%). The fact that HL3/5 did not possess a generally higher ability to penetrate successfully to form haustoria on the susceptible barley line, indicates that HL3/5 did not overcome the mlo5 resistance by being generally more vigorous. In the resistant barley line, papillae were larger than in the susceptible line; however, both isolates were associated with papillae of the same diameter at the time of penetration. We suggest that the mlo5 resistant barley line confers two different forms of resistance: isolate-specific and isolate-nonspecific.     

Histological Characterization of Resistance to Uromyces viciae-fabae in Faba Bean

ABSTRACT Components of resistance to the faba bean rust (Uromyces viciae-fabae) were studied at the histological level in seedlings and adult plants of nine faba bean (Vicia faba) lines differing in their level of resistance. Resistance of these lines was previously shown to be characterized macroscopically by an increased latent period, a decreased colony size, and a relatively decreased infection frequency. In some lines, the resistance also was associated with macroscopically visible necrosis. Histological investigations revealed few differences in spore germination and appressorium formation. Significant levels of aborted stomatal penetration by the rust fungus were found on all resistant lines. However, differences among lines were more evident once the stomata were penetrated by the infection structures. Resistance was mainly due to a restriction of haustorium formation with varying levels of early abortion of the colonies, a reduction in the number of haustoria per colony, and smaller colony size. In addition, necrosis of the host cells associated with infection hyphae was detectable in some lines from the beginning of colony development. This microscopically visible necrosis became stronger from 4 days after inoculation, resulting in a reduced growth of the colony. Differences in resistance levels were more marked in adult plants than in seedlings.     

欧亚种葡萄自交F1代对白粉病和霜霉病的抗性遗传

采用田间自然鉴定、田间接种鉴定及室内离体叶圆片接种鉴定三种方法,研究了10个欧亚种葡萄品种(系)、一个欧山杂种及其493株自交后代幼苗对葡萄霜霉病及白粉病的抗性及遗传关系,同时对其自交后代抗白粉病和霜霉病的遗传趋势进行了分析.结果表明,供试品种(系)及其自交后代对霜霉病与白粉病的抗性存在极显著相关,自交后代对两病表现伴随遗传现象,初步推断这可能是由葡萄抗病基因的多效性引起.在欧亚种葡萄品种自交后代群体中,虽然90%以上都是感病性中等或高的类型,但仍能够得到一定比例(10%以下)抗病性强的类型,这为利用欧亚种葡萄品种进行自交或品种间杂交选育优质抗病新品种提供了依据.     

Identification of race-specific resistance in North American Vitis spp. limiting Erysiphe necator hyphal growth

Race-specific resistance against powdery mildews is well documented in small grains but, in other crops such as grapevine, controlled analysis of host-pathogen interactions on resistant plants is uncommon. In the current study, we attempted to confirm powdery mildew resistance phenotypes through vineyard, greenhouse, and in vitro inoculations for test cross-mapping populations for two resistance sources: (i) a complex hybrid breeding line, 'Bloodworth 81-107-11', of at least Vitis rotundifolia, V. vinifera, V. berlandieri, V. rupestris, V. labrusca, and V. aestivalis background; and (ii) Vitis hybrid 'Tamiami' of V. aestivalis and V. vinifera origin. Statistical analysis of vineyard resistance data suggested the segregation of two and three race-specific resistance genes from the two sources, respectively. However, in each population, some resistant progeny were susceptible in greenhouse or in vitro screens, which suggested the presence of Erysiphe necator isolates virulent on progeny segregating for one or more resistance genes. Controlled inoculation of resistant and susceptible progeny with a diverse set of E. necator isolates clearly demonstrated the presence of fungal races differentially interacting with race-specific resistance genes, providing proof of race specificity in the grape powdery mildew pathosystem. Consistent with known race-specific resistance mechanisms, both resistance sources were characterized by programmed cell death of host epidermal cells under appressoria, which arrested or slowed hyphal growth; this response was also accompanied by collapse of conidia, germ tubes, appressoria, and secondary hyphae. The observation of prevalent isolates virulent on progeny with multiple race-specific resistance genes before resistance gene deployment has implications for grape breeding strategies. We suggest that grape breeders should characterize the mechanisms of resistance and pyramid multiple resistance genes with different mechanisms for improved durability.     

Osmotin and thaumatin from grape: a putative general defense mechanism against pathogenic fungi

ABSTRACT Little information is available concerning the expression of pathogenesis-related (PR) proteins in grapevine (Vitis vinifera) and their effect properties on the major fungal pathogens of grape. A systematic study was performed on the effect of total or individual grape proteins on mycelial growth, spore germination, and germ tube growth of Uncinula necator, Phomopsis viticola, and Botrytis cinerea. Two proteins, identified as PR proteins by immunological methods and by N-terminal sequencing as osmotin and thaumatin-like protein, exhibited strong antifungal activities in vitro, blocking the growth of Phomopsis viticola and Botrytis cinerea mycelia. In addition, they inhibited spore germination and germ tube growth of U. necator, Phomopsis viticola, and Botrytis cinerea. The presence of both proteins displayed a synergistic effect. The expression of osmotin and thaumatin-like protein was induced in grapevine leaves and berries infected with U. necator and Phomopsis viticola. Thaumatin previously was thought to occur exclusively in berries. Immunoblot analyses revealed the accumulation of the two PR proteins in infected leaves and berries, supporting a role in vivo in increasing the resistance of grapevine to fungal attack.     

Infection of canola by secondary zoospores of Plasmodiophora brassicae produced on a nonhost

Clubroot, caused by Plasmodiophora brassicae, has two infection stages (primary and secondary). Although primary infection occurs in many plant species, secondary infection only continues to completion in susceptible hosts. As part of a larger study of clubroot pathogenesis, secondary zoospores collected from infected root hairs of canola and ryegrass were inoculated onto healthy roots of both plant species. The treatments consisted of all possible combinations of the two plant species and the two sources of inoculum. At 5 days after inoculation, levels of root hair infection were similar and in a range of 50–68% on roots in all of the treatments. Secondary infection was also observed from all of the treatments, with approximately 50% on canola and 40% on ryegrass. The proportion of secondary infection and the number of secondary plasmodia were higher in canola inoculated with zoospores from canola than in ryegrass inoculated with zoospores from ryegrass, with the other combinations intermediate. At 35 days after inoculation, typical clubs developed on 14% of the canola plants inoculated with secondary zoospores from canola, and tiny clubs developed on 16% of the canola plants inoculated with zoospores from ryegrass. Secondary infection occurred in about one-third of ryegrass plants but no clubs developed, regardless of inoculum source. These results indicate that resistance to secondary infection in ryegrass is induced during primary infection. This is the first report that secondary zoospores produced on a nonhost can infect a host and reconfirms that secondary infection can occur in a nonhost.     

Correlations of Morphological, Anatomical, and Chemical Features of Grape Berries with Resistance to Botrytis cinerea

ABSTRACT Resistance of mature berries of grapevine cultivars and selections to postharvest infection by Botrytis cinerea was assessed. Little or no resistance existed in most popular table grape Vitis vinifera cultivars, except in moderately resistant 'Emperor' and 'Autumn Black'. Highly resistant grapes were V. rotundifolia, V. labrusca, or other complex hybrids. Morphological, anatomical, and chemical characteristics of 42 genetically diverse cultivars and selections with various levels of resistance to B. cinerea were examined to determine which features were associated with resistance. We quantified the (i) density of berries within a cluster; (ii) number of pores and lenticels on the berry surface; (iii) thickness and number of cell layers in the epidermis and external hypodermis; (iv) amount of cuticle and wax; (v) berry skin protein content; (vi) total phenolic content of the skin before and after B. cinerea inoculation; and (vii) catechin and trans- and cis-resveratrol contents of the skin before and after inoculation. The number of pores was negatively correlated with resistance. Highly resistant cultivars had few or no pores in the berry surface. The number and thickness of epidermal and hypodermal cell layers and cuticle and wax contents were positively correlated with resistance. Other characteristics evaluated were not associated with resistance. trans-Resveratrol and cis-resveratrol were induced by B. cinerea inoculation only in sensitive and moderately resistant cultivars and selections.     

Ontogenic resistance and plant disease management: a case study of grape powdery mildew

ABSTRACT A fundamental principle of integrated pest management is that actions taken to manage disease should be commensurate with the risk of infection and loss. One of the less-studied factors that determines this risk is ontogenic, or age-related resistance of the host. Ontogenic resistance may operate at the whole plant level or in specific organs or tissues. Until recently, grape berries were thought to remain susceptible to powdery mildew (Uncinula necator) until late in their development. However, the development of ontogenic resistance is actually quite rapid in berries, and fruit become nearly immune to infection within 4 weeks after fruit set. Our objective was to determine how and at what stage the pathogen was halted in the infection process on ontogenically resistant berries. Adhesion of conidia, germination, and appressorium formation were not impeded on older berries. However, once berries were approximately 3 weeks old and older, few germlings were able to form secondary hyphae. Ontogenically resistant berries responded rapidly to infection by synthesis of a germin-like protein that had been previously shown to play a role in host defense against barley powdery mildew. On susceptible berries, cell discoloration around penetration sites indicated the oxidation of phenolic compounds; a process that was followed by localized cell death. However, the pathogen was still able to infect such cells prior to their death, continue secondary growth, and thereby colonize young berries. Formation of papillae was not involved in the differential resistance mechanism of older berries. In susceptible berries, papillae formed frequently at infection sites but did not always contain the pathogen, whereas in resistant berries, the pathogen was always halted prior to the formation of papillae. The host defense, which conditions ontogenic resistance, operates in the earliest stages of the infection process, in the absence of gross anatomical barriers, prior to the formation of a functional haustorium and prior to the development of a conspicuous penetration pore. We also found that diffuse powdery mildew colonies that were not visible in the field predisposed berries to bunch rot by Botrytis cinerea, increased the levels of infestation by spoilage microorganisms, and substantially degraded wine quality. Our improved understanding of the nature, causes, and stability of ontogenic resistance in the grapevine/ powdery mildew system has supported substantial changes in how fungicides are used to control the disease. Present applications are more focused on the period of maximum fruit susceptibility instead of following a calendar-based schedule. This has improved control, reduced losses, and in many cases reduced the number of fungicide applications required to suppress the disease. Particularly where fungicides are deployed in a programmatic fashion and ontogenic resistance is dynamic, there may be equivalent improvements to be made in other hostpathogen systems through studies of how host susceptibility changes through time.