Measures of durability of resistance

ABSTRACT Conventional models for the durability of resistant cultivars focus on the dynamics of the frequency of resistance genes. This leads to a definition of the durability of resistance as the time from introduction of the cultivar to the time when the frequency of the virulence gene reaches a preset threshold. It is questionable whether this is the most appropriate way to measure durability. Here we use a simple epidemiological model to link population dynamics and population genetics to compare three measures of durability: (i) the expected time until invasion of the virulent genotype, by mutation or immigration, and subsequent establishment of a population (T(invasion)); (ii) the virulence frequency related measure of the time for the virulent genotype to take-over the pathogen population ( T(take-over)); and (iii) the additional yield, measured by the additional number of uninfected host growth days (T(additional)). Specifically, we show how the measures of durability are affected by deployment and epidemiological parameters. We use a combination of numerical solution and analytical approximation of a model for the population dynamics of avirulent and virulent genotypes of a pathogen growing in dynamically changing populations of resistant and susceptible cultivars. The three measures of durability are compared. Some consequences of the results for durable resistance in multilines and mixtures and the regional deployment of resistant cultivars are discussed.     

Selection of virulent populations of Meloidogyne javanica by repeated cultivation of Mi resistance gene tomato rootstocks under field conditions

Field trials were conducted in a plastic house artificially infested with an avirulent population of Meloidogyne javanica to determine the durability of the resistance mediated by the Mi gene in tomato rootstocks after repeated cultivation for three consecutive years. Treatments included an experimental rootstock cv. PG76 ( Solanum lycopersicum  ×  Solanum sp.), a commercial rootstock cv. Brigeor ( S. lycopersicum  ×  S. habrochaites ), a resistant tomato cv. Monika ( S. lycopersicum ), and a susceptible cv. Durinta ( S. lycopersicum ). Based on the reproduction index (RI: number of eggs per g root on the resistant cultivar divided by number of eggs per g root on the susceptible cultivar × 100), rootstock cv. PG76 responded as highly resistant (RI = 7%) after the first cropping cycle (3·4 nematode generations), showed intermediated resistance (RI = 33%) after the second cropping cycle (3·3 generations), and was fully susceptible (RI = 94%) after the third cycle (3·3 generations). In contrast, rootstock cv. Brigeor and resistant cv. Monika retained intermediate resistance levels (RI = 41 and 25%, respectively) after the third cropping cycle. Virulent nematode populations were rapidly selected from an avirulent one after repeated cultivation of resistant tomatoes under field conditions. Bioassays conducted under controlled conditions confirmed that selection for virulence occurred more rapidly in plots with cv. PG76 followed by Brigeor and Monika. The nematode population in the field not exposed to Mi resistance remained avirulent to Mi genotypes. The genetic background of the resistant rootstocks and the frequency of cropping were critical factors for the appearance of virulent nematode populations. Irrespective of nematode infection, all resistant tomatoes yielded more than the susceptible cultivar.     

Efficacy of the R2 resistance gene as a component for the durable management of potato late blight in France

Many race-specific resistance genes to potato late blight are overcome in France, but the disease appears later on genotypes carrying the R2 gene. This study examined whether R2 could contribute to durable late-blight control in France, and analysed the conditions of its performance. Plants grown from tubers of different physiological ages showed no difference in R2 expression in field and climate-chamber experiments, demonstrating that the delay in epidemic onset provided by R2 was not the result of gene inactivation in old plants. Among isolates collected at one site, those virulent on R2 were classified into three AFLP profiles. AFLP-VII comprised exclusively isolates virulent to R2 , whereas AFLP-IV and AFLP-V included both virulent and avirulent isolates. No significant aggressiveness differences were observed between virulent and avirulent isolates from AFLP-V; however, isolates from AFLP-VII were significantly less aggressive than virulent isolates from AFLP-V. These results indicate that: (i) the delayed onset of epidemics on R2 cultivars is the result of the breakdown of R2 by virulent isolates; (ii) aggressiveness of isolates virulent to R2 depends primarily on the genetic background of the pathogen where the mutation to virulence occurs; and (iii) this mutation does not lead per se to lower pathogenic fitness. It is suggested that R2 is unlikely to make a lasting contribution to late-blight control in France, and that diversification strategies such as cultivar mixtures might not considerably increase its durability.     

An integrated multivariate approach to net blotch of barley: Virulence quantification,pathotyping and a breeding strategy for disease resistance

Knowledge of pathotype diversity and virulence in local populations of Pyrenophora teres is a prerequisite to screening for durable resistance to net blotch. The current study aimed to quantify the virulence level of Moroccan isolates, identify and designate existing pathotypes, and select resistant genotypes. We developed a method for virulence quantification of P. teres isolates based on a conversion of infection responses into frequencies for use in correspondence analysis. Coordinates of the first axis of this analysis had a virulence spectrum and ranked isolates from virulent to avirulent. Mixed model analysis was also devised for virulence quantification. Coordinates of the first dimension of correspondence analysis were linearly correlated to BLUPs (Best Linear Unbiased Predictors) of the mixed model. A genotype by genotype by environment model (GGE) coupled with cluster analysis differentiated P. teres isolates into ten and nine pathotypes for net- and spot-forms respectively. Populations of these two forms were dissimilar in terms of classes of virulence. For P. teres f. maculata, avirulent, moderately virulent and highly virulent isolates represented one-third of the population, whereas 90% of P. teres f. teres population was composed of avirulent to moderately avirulent isolates. Barley differential sets were subsequently reduced to two new sets that simplified pathotyping through a key code based on resistant or susceptible reactions. Dendrograms of cluster analysis based on GGE analysis depicted the stability of a genotype’s reactions across all isolates, and using only resistant cultivars as sources of resistance to control net blotch disease would, based on this analysis, fail to control all pathotypes. Therefore, we propose an alternative breeding strategy to control net blotch effectively.     

The impact of wheat cultivar mixtures on virulence dynamics in Zymoseptoria tritici populations persists after interseason sexual reproduction

This study follows on from a previous study showing that binary mixtures of wheat cultivars affect the evolution of Zymoseptoria tritici populations within a field epidemic from the beginning (t1) to the end (t2) of a growing season. Here, we focused on the impact of interseason sexual reproduction on this evolution. We studied mixtures of susceptible and resistant cultivars (carrying Stb16q, a recently broken-down resistance gene) in proportions of 0.25, 0.5 and 0.75, and their pure stands. We determined the virulence status of 1440 ascospore-derived strains collected from each cultivar residue by phenotyping on seedlings. Virulence frequencies in the ascospore-derived population were lower in mixtures than in pure stands of the resistant cultivar, especially in the susceptible cultivar residues, as at t2, revealing that the impact of mixtures persisted until the next epidemic season (t3). Surprisingly, after sexual reproduction the avirulence frequencies on the resistant cultivar residues increased in mixtures where the proportion of the susceptible cultivar was higher. Our findings highlight two epidemiological processes: selection within the pathogen population between t1 and t2 driven by asexual cross-contamination between cultivars (previous study) and sexual crosses between avirulent and virulent strains between t2 and t3 driven by changes in the probabilities of physical encounters (this study). Mixtures therefore appear to be a promising strategy for the deployment of qualitative resistances, not only to limit the intensity of Septoria tritici blotch epidemics, but also to reduce the erosion of resistances by managing evolution of the pathogen population at a pluriannual scale.     

The reproductive potential of the root-knot nematode <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> is affected by selection for virulence against major resistance genes from tomato and pepper

The emergence of virulent root-knot nematode populations, able to overcome the resistance conferred by some of the resistance genes (R-genes) in Solanaceous crops, i.e., Mi(s) in tomato, Me(s) in pepper, may constitute a severe limitation to their use in the field. Research has been conducted to evaluate the durability of these R-genes, by comparing the reproduction of several laboratory-selected and wild virulent Meloidogyne incognita isolates, on both susceptible and resistant tomatoes and peppers. We first show that the Me1 R-gene in pepper behaves as a robust R-gene controlling avirulent and virulent Me3, Me7 or Mi-1 isolates. Although the reproductive potential of the virulent isolates was highly variable on susceptible and resistant plants, we also confirm that virulence is highly specific to a determined R-gene on which selection has occurred. Another significant experimental result is the observation that a reproductive fitness cost is associated with nematode virulence against Mi-1 in tomato and Me3 and Me7 in pepper. The adaptative significance of trade-offs between selected characters and fitness-related traits, suggests that, although the resistance can be broken, it may be preserved in some conditions if the virulent nematodes are counter-selected in susceptible plants. All these results have important consequences for the management of plant resistance in the field.     

Durability of natural and transgenic resistances in rice to <Emphasis Type="Italic">Rice yellow mottle virus</Emphasis>

A monogenic recessive resistance to Rice yellow mottle virus (RYMV) found in the Oryza sativa indica cultivar Gigante and in a few Oryza glaberrima cultivars provided a higher level of resistance than either a polygenic partial resistance found in some japonica cultivars which delayed symptom expression or transgenic resistances which were partial and temporary. This high resistance was overcome by several isolates, but the percentage of such virulent isolates in the fields was low. There was no relationship between the virulence of an isolate towards the high resistance and its aggressiveness in other cultivars. Isolates with either of the two components of pathogenicity – virulence and aggressiveness – were found in each strain and in all regions of Africa, in both wild and cultivated grass species. There was no loss of fitness of resistance-breaking (RB) isolates as they were not counter-selected, impaired or outperformed after serial passages in susceptible cultivars, even in mixture with avirulent quasi-isogenic wild type isolates. Resistance breaking was highly dependent on the amount of virus inoculated and on the mode of transmission. Implications of these results for the durability of the resistances to RYMV and for the development of integrated disease management strategies are discussed.     

Lack of influence of host plant disease resistance on the evolution of resistance to sterol demethylation-inhibiting (DMI) fungicides in<Emphasis Type="Italic">Cercospora beticola</Emphasis>

Field experiments were conducted during 1997 and 1998 to determine the effects of sugar beet cultivar susceptibility to Cercospora leaf-spot on the sensitivity ofCercospora beticola isolates to the triazole fungicide flutriafol. Four cultivars with different levels of disease resistance were treated in experimental plots with six spray applications of flutriafol. Disease assessments were carried out at 15-day intervals. Sensitivity to flutriafol was measured on isolates collected from the plots ∼15 days after the last flutriafol application. Measurements of disease severity and calculations of AUDPC (area under disease progress curve) values showed a distinct differentiation among cultivars, reflecting their level of disease resistance. Disease severity was significantly lower in cvs. ‘Bianca’ and ‘Areth’ than in ‘Univers’ and ‘Rizor’ both in the untreated and in the flutriafol-treated plots. Fungal isolates from flutriafol-treated plots were less sensitive to the fungicide than were isolates from untreated plots. However, no differences in isolate sensitivity were observed among the cultivars, as regards their level of disease resistance. Despite the fact that the use of resistant cultivars cannot eliminate selectively the resistant strains, it can eliminate both resistant and sensitive isolates. Reducing the number of treatments with DMIs, by applying them only when environmental conditions are favorable for disease development, is a prerequisite for successful resistance management; therefore, the use of disease-resistant varieties could aid toward management of DMIs resistance inC. beticola. http://www.phytoparasitica.org posting May 6, 2003.     

Annual dynamics of Zymoseptoria tritici populations in wheat cultivar mixtures: A compromise between the efficacy and durability of a recently broken-down resistance gene?

Cultivar mixtures slow polycyclic epidemics but may also affect the evolution of pathogen populations by diversifying the selection pressures exerted by their plant hosts at field scale. We compared the dynamics of natural populations of the fungal pathogen Zymoseptoria tritici in pure stands and in three binary mixtures of wheat cultivars (one susceptible cultivar and one cultivar carrying the recently broken-down Stb16q gene) over two annual field epidemics. We combined analyses of population “size” based on disease severity, and of population “composition” based on changes in the frequency of virulence against Stb16q in seedling assays with more than 3000 strains. Disease reductions were observed in mixtures late in the epidemic, at the whole-canopy scale and on both cultivars, suggesting the existence of a reciprocal protective effect. The three cultivar proportions in the mixtures (0.25, 0.5, and 0.75) modulated the decrease in (a) the size of the pathogen population relative to the two pure stands, (b) the size of the virulent subpopulation, and (c) the frequency of virulence relative to the pure stand of the cultivar carrying Stb16q. Our findings suggest that optimal proportions may differ slightly between the three indicators considered. We argue potential trade-offs that should be taken into account when deploying a resistance gene in cultivar mixtures: between the dual objectives “efficacy” and “durability,” and between the “size” and “frequency” of the virulent subpopulation. Based on current knowledge, it remains unclear whether virulent subpopulation size or frequency has the largest influence on interepidemic virulence transmission.     

Effectiveness and profitability of the <Emphasis Type="Italic">Mi</Emphasis>-resistant tomatoes to control root-knot nematodes

Experiments were conducted to determine the effectiveness and profitability of the Mi-resistance gene in tomato in suppressing populations of Meloidogyne javanica in a plastic-house with a natural infestation of the nematode. Experiments were also conducted to test for virulence and durability of the resistance. Monika (Mi-gene resistant) and Durinta (susceptible) tomato cultivars were cropped for three consecutive seasons in non-fumigated or in soil fumigated with methyl bromide at 75 g m^–2 and at a cost of 2.44 euros m^–2. Nematode densities were determined at the beginning and end of each crop. Yield was assessed in eight plants per plot weekly for 6 weeks. The P_f/P_i values were 0.28 and 21.6 after three crops of resistant or susceptible cultivars, respectively. Growth of resistant as opposed to susceptible tomato cultivars in non-fumigated soil increased profits by 30,000 euros ha^–1. The resistant Monika in non-fumigated soil yielded similarly (P > 0.05) to the susceptible Durinta in methyl bromide fumigated soil but the resistant tomato provided a benefit of 8800 euros ha^–1 over the susceptible one because of the cost of fumigation. Selection for virulence did not occur, although the nematode population subjected to the resistant cultivar for three consecutive seasons produced four times more eggs than the population on the susceptible one. Such a difference was also shown when the resistant cultivar was subjected to high continuous inoculum pressure for 14 weeks. The Mi-resistance gene can be an effective and economic alternative to methyl bromide in plastic-houses infested with root-knot nematodes, but should be used in an integrated management context to preserve its durability and prevent the selection of virulent populations due to variability in isolate reproduction and environmental conditions.     

Rapid screening of <Emphasis Type="Italic">Musa</Emphasis> species for resistance to Fusarium wilt in an <Emphasis Type="Italic">in vitro</Emphasis> bioassay

In order to accelerate breeding and selection for disease resistance to Fusarium wilt, it is important to develop bioassays which can differentiate between resistant and susceptible cultivars efficiently. Currently, the most commonly used early bioassay for screening Musa genotypes against Fusarium oxysporum f. sp. cubense (Foc) is a pot system, followed by a hydroponic system. This paper investigated the utility of in vitro inoculation of rooted banana plantlets grown on modified medium as a reliable and rapid bioassay for resistance to Foc. Using a scale of 0 to 6 for disease severity measurement, the mean final disease severities of cultivars expressing different levels of disease reaction were significantly different (P ≤ 0.05). Twenty-four days after inoculation with Foc tropical race 4 at 10⁶ conidia ml⁻¹, the plantlets of two susceptible cultivars had higher final disease severities than that of four resistant cultivars. Compared with ‘Guangfen No.1’, ‘Brazil Xiangjiao’ is highly susceptible to tropical race 4 and its mean final disease severity was the highest (5.27). The plantlets of moderately resistant cultivar ‘Formosana’ had a mean final disease severity (3.53) lower than that of ‘Guangfen No.1’ (4.33) but higher than that of resistant cultivars: ‘Nongke No.1’, GCTCV-119, and ‘Dongguan Dajiao’ (1.87, 1.73, and1.53, respectively). Promising resistant clones acquired through non-conventional breeding techniques such as in vitro selection, genetic transformation, and protoplast fusion could be screened by the in vitro bioassay directly. Since there is no acclimatization stage for plantlets used in the bioassay, it helps to improve banana breeding efficiency.     

Physiologic races ofFusarium oxysporum f.sp.melonis in the southeastern anatolia region of turkey and varietal reactions to races of the pathogen

Thirty-four isolates ofFusarium oxysporum f.sp.melonis (F.o.m.) obtained from 205 fields in melon-producing areas in the southeastern Anatolia Region of Turkey were identified on the basis of colony morphology and pathogenicity by the root dip method. In this region the mean prevalence of wilt disease was 88.1% and the mean incidence of disease was 47.5%. Physiologic races 0, 1, 2, and 1,2 of the pathogen were determined by their reactions on differential melon cultivars ‘Charentais T,’ ‘Isoblon’, ‘Isovac’ and ‘Margot’ in the greenhouse. Race 1,2, representating 58.8% (20/34) of all isolates, was widely distributed. Of the other pathogenic isolates, eight were identified as race 0, five as race 1, and one as race 2. This is the first report of physiologic races ofF.o.m. in Turkey. Of 44 melon cultivars tested in the greenhouse for resistance toF.o.m. races, 36 were found to be moderately resistant to race 0, 17 were susceptible to race 1,2, 34.1% were highly resistant to race 1, and 52.2% had moderate resistance to race 2. http://www.phytoparasitica.org posting July 16, 2002.     

Induction of a defense response in strawberry mediated by an avirulent strain of <Emphasis Type="Italic">Colletotrichum</Emphasis>

In the strawberry crop area of Tucumán (north-west Argentina) the three species of Colletotrichum causing anthracnose disease (C. acutatum, C. fragariae and C. gloeosporioides) were detected. Among all isolates characterized, one of them identified as C. acutatum (M11) and another as C. fragariae (F7) were selected due to their conspicuous interaction with the strawberry cultivar Pájaro. Whereas isolate M11 produced a strong compatible interaction in cv. Pájaro with clear disease symptoms (DSR = 5.0), the isolate F7 brought about a typical incompatible interaction (DSR = 1.0). When plants of cv. Pájaro were inoculated with F7 prior to the inoculation with M11, the former avirulent strain prevented the growth of the latter virulent pathogen. Experimental evidence indicated that the time elapsed between the first inoculation with the avirulent pathogen and the second inoculation with the virulent one was crucial to inhibit the growth of the latter. The growth of F7 on the plant without provoking damage and the fact that there was no in vitro antagonistic effect between the pathogens, suggests that the avirulent strain triggers a plant defensive response against M11. The defense response was further confirmed by the detection of an early oxidative burst occurring within 4 h after the first inoculation and by the observation of anatomical changes associated with defense mechanisms that lasted 50 days after the inoculation with F7. Results obtained support the hypothesis that the plant resistance against the virulent strain M11 is elicited by one or more diffusible(s) compound(s) produced by the avirulent strain F7.     

Aggressiveness of eight Venturia inaequalis isolates virulent or avirulent to the major resistance gene Rvi6 on a non‐Rvi6 apple cultivar

For sustainable management of scab‐resistant apple cultivars, it is necessary to understand the role of aggressiveness in the adaptation of Venturia inaequalis populations and particularly the costs to the organism of acquiring additional virulence. The aims of the present study were (i) to identify the quantitative variables that are most important in determining the differences in aggressiveness among groups of V. inaequalis isolates, and (ii) to ascertain whether virulent and avirulent isolates of V. inaequalis differ significantly in aggressiveness. The aggressiveness of eight isolates that differed in their virulence to the major resistance gene Rvi6 was compared on the non‐Rvi6 apple cv. Gala. Three components of aggressiveness, namely lesion density, the number of spores per square centimetre of leaf area, and the number of spores per lesion, were evaluated 21 days after inoculation, and the kinetics of lesion density over time were analysed in terms of maximum lesion density, length of latent period and rate of lesion appearance. On the second youngest but fully developed leaf at the time of inoculation, maximum lesion density in the virulent group was 20% lower and the latent period 7% longer, than in the avirulent group. However, the alternative hypothesis, namely that isolates had adapted to quantitative resistance present in cv. Gala depending on their cultivar of origin, could not be rejected. The analysis of the kinetics of lesion density by a non‐linear mixed‐effect model proved useful in the assessment of aggressiveness.     

Histochemical and chemical evidence for lignin accumulation during the expression of resistance to leaf rust fungi in wheat

Levels of individual phenolic acids were examined in primary leaves of wheat (Triticum aestivum) after inoculation with avirulent and virulent strains of the leaf rust fungus (Puccinia recondita f. sp. tritici) at stages when previous work had shown fungal and host cells to be affected by expression of the Lr20 or Lr28 alleles for resistance. The predominant phenolic acid, ferulic acid, as well as p-coumaric and syringic acids were detected in primary leaves in both unbound and bound forms. They were not detected in germinating urediniospores of either rust strain. Levels of unbound phenolic acids changed little in response to infection. In Lr28-bearing leaves inoculated with an avirulent strain, increased concentrations of bound phenolic acids and three other unidentified compounds were observed about 4 h after many single or small groups of cells had undergone hypersensitive collapse. In an Lr20-bearing cultivar, levels of bound phenolic acids fell in leaves inoculated with either a virulent or avirulent rust strain. Coniferyl aldehyde and coniferyl alcohol were not detected in healthy or inoculated leaves of either wheat cultivar. Attempts to affect expression of resistance by application of inhibitors of phenylalanine ammonia-lyase were not successful and both wheat cultivars remained resistant to avirulent rust strains. The bound phenolic acids which accumulate in cells undergoing a hypersensitive response may play a role in resistance of Lr28-bearing wheat to the leaf rust fungus.     

Resistance to fusarium basal rot of onion in greenhouse and field and associated expression of antifungal compounds

Greenhouse and field evaluations of onion for resistance to Fusarium basal rot caused byFusarium oxysporum f.sp.cepae were conducted on cultivars ‘Akgün 12’ and ‘Rossa Savonese’ previously described as resistant at the seedling stage. In the greenhouse experiments inoculations were carried out on seeds or soil; in the field experiments evaluation was performed on onion sets from plants grown in naturally infested soils. Akgün 12 and to a lesser extent Rossa Savonese were resistant to the disease at the bulb stage in all experiments. Results were also consistent with those obtained from a previous screening at the seedling stage. Onion sets were also extracted and fractionated by thin layer chromatography to determine their content of antifungal compounds. Extracts were characterized by the expression of distinct antifungal components, which may be involved in resistance to the pathogen. http://www.phytoparasitica.org posting July 14, 2004.     

Dissemination of Information About Management Strategies and Changes in Farming Practices for the Exploitation of Resistance to Leptosphaeria maculans (Phoma Stem Canker) in Oilseed Rape Cultivars

The management of phoma stem canker (blackleg disease, caused by Leptosphaeria maculans) is an integral component of oilseed rape production. In this paper, we discuss the information about management strategies that is disseminated in Europe and Australia. New cultivars have been introduced with improved resistance to disease, but sometimes this resistance has been overcome as new races of the pathogen have emerged. When cultivars with single major gene resistance have been introduced into areas with high inoculum concentrations, significant economic damage has been caused by new races of L. maculans within 2–3 years. Quantitative or polygenic resistance has also been used successfully against stem canker and offers more durable disease resistance if plant breeders and farmers deploy this resistance more effectively. Strategies to improve the durability of resistance need to be developed and tested in practice. New information on the occurrence of virulence and avirulence genes in populations of Leptosphaeria maculans and modelling of the durability of resistance provide opportunities for plant breeders, specialist technical organisations, cooperatives, advisory services and farmers to collaborate and better exploit cultivar resistance. Changing economic and environmental factors influence cropping practices and, if to be considered successful, management strategies must show clear financial benefits. Technology transfer will need to address all aspects of managing stem canker and other diseases of oilseed rape and using effective written, verbal and electronic methods of communication.     

Resistance to <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> expresses a hypersensitive-like response in <Emphasis Type="Italic">Coffea arabica</Emphasis>

Root-knot nematodes (RKN) are obligate parasite species of the genus Meloidogyne that cause great losses in Arabica coffee (Coffea arabica L.) plantations. Identification of resistant genotypes would facilitate the improvement of coffee varieties aiming at an environmental friendly and costless nematode control. In this work, the C. arabica genotype ‘UFV 408-28’ was found to be resistant to the most destructive RKN species M. incognita. Pathogenicity assays indicated that the highly aggressive populations of M. incognita races 1, 2 and 3 were not able to successfully reproduce on ‘UFV 408-28’ roots and displayed a low gall index (GI = 2). An average reduction of 87% reduction of the M. incognita population was observed on ‘UFV 408-28’ when compared to the susceptible cultivar ‘IAC 15’. By contrast, ‘UFV 408-28’ was susceptible to the related species M. exigua and M. paranaensis (GI = 5 and 4, respectively). Histological observations performed on sections of UFV408-28 roots infected with M. incognita race 1 showed that nematode infection could be blocked right after penetration or during migration and establishment stages, at 6 days, 7 days and 8 days after infection (DAI). Fluorescence and bright field microscopy observations showed that root cells surrounding the nematodes exhibited HR-like features such as accumulation of phenolic compounds and a necrotic cell aspect. In the susceptible ‘IAC 15’ roots, 6 DAI, feeding sites contained giant cells with a dense cytoplasm. Necrotic cells were never observed throughout the entire infection cycle. The HR-like phenotype observed in the ‘UFV 408-28’—M. incognita interaction suggests that the coffee resistance may be mediated by a R-gene based immunity system and may therefore provide new insights for understanding the molecular basis of RKN resistance in perennial crops.     

Molecular and biological characterization of <Emphasis Type="Italic">Chrysanthemum stem necrosis virus</Emphasis> isolates from distinct regions in Japan

Three isolates of Chrysanthemum stem necrosis virus (CSNV) were obtained from chrysanthemum plants in distinct regions of Japan in 2006 and 2007. All the original host plants showed severe necrotic symptoms on the leaves and stems. Amino acid sequence data of the nucleocapsid protein genes of the three isolates (CbCh07A, TcCh07A, and GnCh07S) showed high identities with those of two other CSNV isolates, HiCh06A L1 from Japan and Chry1 from Brazil. Furthermore, for the first time the complete nucleotide sequence of the S RNA was determined for CSNV (isolate HiCh06A). In phylogenetic analysis based on the non-structural protein genes from the genus Tospovirus, HiCh06A L1 was placed in the same genetic group as Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus. Host range examination for isolates HiCh06A L1 and CbCh07A showed that green pepper (cv. ‘Kyoyutaka’, ‘Saitamawase’, ‘Tosakatsura’, ‘L3 sarara’ and ‘L3 miogi’) and tomato (cv. ‘Sekaiichitomato’) were systemically susceptible hosts, whereas TSWV-resistant Solanaceae species, Capsicum chinense, Lycopersicon peruvianum and a TSWV-resistant cultivar of green pepper (cv. TSR miogi), were resistant.     

Host genotype- and temperature-dependent colonizationof In vitro carnation callus cultures byFusarium oxysporum f.sp.dianthi race 2

Differentin vivo resistance/susceptibility levels of 14 carnation cultivars toFusarium oxysporum f.sp.dianthi race 2, the causal agent of Fusarium wilt disease of carnation, were also expressed in anin vitro system and assayed as the degree of fungal colonization of callus cultures at 20° C. Temperature influenced thein vitro expression of carnation resistance. An incubation temperature of 27° C increased the colonization of calli derived from both the susceptible (‘Corrida’ and ‘Ambra’) and the resistant (‘Pulcino’ and ‘Pallas’) cultivars. At 15°C, the colonization of calli derived from Pulcino and Pallas diminished significantly more than for Ambra and Corrida. Inhibition of fungal growth on resistant calli was correlated to retardation in hyphal development. Both scanning electron microscopy and light microscopy observations showed that hyphae did not penetrate into carnation cells.