Development and Evaluation of an in vitro Detached Leaf Assay forc Pre-Screening Resistance to Fusarium Head Blight in Wheat

An in vitro detached leaf assay, involving the inoculation of detached leaves with Microdochium nivale, was further developed and used to compare with whole plant resistance ratings to Fusarium head blight (FHB) of 22 commercial cultivars and published information on 21 wheat genotypes, identified as potential sources for FHB resistance. An incubation temperature of 10 °C and isolates of M. nivale var. majus of intermediate pathogenicity were found to be the most suitable for the differential expression of several components of partial disease resistance (PDR), namely incubation period, latent period and lesion length, in wheat genotypes used in the detached leaf assay. There were highly significant differences (P < 0.001) for each component of PDR within commercial cultivars and CIMMYT genotypes. Positive correlations were found between incubation period and latent period (r = 0.606; P < 0.001 and r = 0.498; P < 0.001, respectively, for commercial cultivars and CIMMYT genotypes), inverse correlations between incubation period and lesion length (r = -0.466; P < 0.01 and r = –0.685; P < 0.001, respectively) and latent period and lesion length (r = –0.825; P < 0.001 and r = –0.848; P < 0.001, respectively). Spearman rank correlations between individual PDR components and UK 2003 recommended list ratings were significant for incubation period (r_s = 0.53; P < 0.05) and latent period (r_s = 0.70; P < 0.01) but not for lesion length (r _s = –0.26). Commercial cultivars identified with high resistances across all three PDR components in the detached leaf assay also had high whole plant FHB resistance ratings, with the exception of cv. Tanker which is more susceptible than the results of the detached leaf assay suggested, indicating an additional susceptibility factor could be present. Agreement between resistances found in the detached leaf assay and resistance to FHB suggests resistances detected in detached leaves are under the same genetic control as much of the resistances expressed in the wheat head of the commercial cultivars evaluated. In contrast, high resistances in each of the PDR components were associated with higher susceptibility across 19 CIMMYT genotypes previously evaluated as potential breeding sources of FHB resistance (incubation period: r = 0.52; P < 0.01, latent period: r = 0.53; P < 0.01, lesion length: r = –0.49; P < 0.01). In particular, the CIMMYT genotypes E2 and E12 together with Summai #3, known to have high levels of whole plant FHB resistance, showed low levels of resistance in each PDR component in the detached leaf assay. Such whole plant resistances, which are highly effective and not detected by the detached leaf assay, do not appear to be present in Irish and UK commercial cultivars. The most resistant Irish and UK commercial cultivars were comparable to the genotype Frontana and the most resistant CIMMYT germplasm evaluated in the leaf assay.     

Detached leaf inoculation of germplasm for rapid screening of resistance to citrus canker and citrus bacterial spot

A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xanthomonas citri subsp. citri, Xcc) and citrus bacterial spot (Xanthomonas alfalfae subsp. citrumelonis, Xac) was developed to evaluate limited quantities of leaf material. Bacterial inocula of Xcc or Xac at 10⁴, 10⁵, or 10⁸ cfu ml⁻¹ were injection-infiltrated into the abaxial surface of disinfested, immature leaves of susceptible and resistant genotypes. Inoculated detached leaves were placed on the surface of 0.5% water agar plates and incubated at 28°C under a 12 h photoperiod. Likewise, inocula were infiltrated into attached leaves of greenhouse plants. At high inoculum concentrations of Xcc or Xac (10⁸ cfu ml⁻¹), resistant cultivars of kumquat developed a hypersensitive-like reaction within 3 days post inoculation (dpi). At 10⁵ cfu ml⁻¹, populations 14 dpi were <10⁴ per inoculation site. In canker-susceptible Citrus spp. (‘Duncan’ grapefruit and ‘Rough’ lemon), water-soaked areas occurred by 3 dpi and typical canker lesions developed by 7 to14 dpi. Concentration of Xcc recovered from inoculation sites was approximately 10⁵ cfu ml⁻¹ by 14 dpi. In citrus bacterial spot-susceptible citrus (‘Swingle’ citrumelo and grapefruit), symptoms developed within 7 dpi. Populations of Xac after inoculation at 10⁵ cfu ml⁻¹ were comparable to Xcc in susceptible hosts 14 dpi (>10⁵). The detached leaf assay is useful for the characterization and differentiation of lesion phenotype for each Xanthomonas pathogen permitting rapid screening of germplasm resistance based on the quantification of number of lesions and bacterial concentration.     

Effect of host factors on the susceptibility of Rhododendron to Phytophthora ramorum

Phytophthora ramorum causes sudden oak death (SOD) in western coastal forests of the USA. In Europe, the pathogen is mainly present in the nursery industry, particularly on Rhododendron. Because of the primary role of Rhododendron as a host and potentially as a vector, the effect of Rhododendron host factors on P. ramorum susceptibility and sporulation was investigated. Inoculation methods using either wounded or non‐wounded detached leaves were applied to 59 Rhododendron cultivars and 22 botanical species, replicated in three separate years. All Rhododendron species and cultivars were susceptible when using wounded leaves, but not when using non‐wounded leaves, suggesting a resistance mechanism operating at the level of leaf penetration. Using a regression tree analysis, the cultivars and species were split into four susceptibility classes. Young leaves were more susceptible than mature leaves when wounded, but less susceptible when non‐wounded. This effect was not correlated with leaf hydrophobicity or the number of leaf hairs. The presence or the type of rootstock did not affect the cultivar susceptibility level. Sporangia and chlamydospore production in the leaf lesions varied widely among Rhododendron cultivars and was not correlated with the susceptibility level. The susceptibility to P. ramorum correlated well with the susceptibility to P. citricola and P. hedraiandra × cactorum, suggesting that the resistance mechanisms against these species are non‐specific. Susceptibility to P. kernoviae was low for most cultivars. These findings have implications for detection, spread and disease control, and are therefore important in pest risk assessment.     

A detached seedling leaf technique to study resistance to Mycosphaerella graminicola (anamorph Septoria tritici) in wheat

A detached seedling leaf technique was developed to screen for resistance to septoria tritici blotch of wheat and to detect specific interactions between cultivars and isolates. Wheat seedlings were inoculated with spore suspensions of Mycosphaerella graminicola . Detached primary leaves were then placed in a clear plastic box such that their cut ends were sandwiched between layers of agar containing benzimidazole, with a gap below the middle of the leaves. Mean levels of disease were affected by light and temperature, and also by the concentration of benzimidazole, such that higher concentrations resulted in less disease. Second leaves were more susceptible than seedling primary leaves. However, none of these factors affected ranking of disease among cultivars or cultivar-by-isolate interactions. Kavkaz–K4500 1.6.a.4, Synthetic 6x and Triticum macha showed specific susceptibility and resistance to different isolates. The detached leaf technique could be a useful complement to field trials and an alternative to whole seedling assays in assessing cultivar resistance and investigating the genetics of the host–pathogen interaction.     

Investigation into components of partial disease resistance,determined <Emphasis Type="Italic">in vitro</Emphasis>, and the concept of types of resistance to <Emphasis Type="Italic">Fusarium</Emphasis> head blight (FHB) in wheat

This work presents an analysis of the relationship between components of partial disease resistance (PDR) detected using in vitro detached leaf and seed germination assays, inoculated with Microdochium majus, and Fusarium head blight (FHB) resistance to Fusarium graminearum assessed using point inoculation, termed Type II resistance. Relationships between in vitro-determined PDR components and FHB resistance using techniques which inoculate the wheat spike uniformly, termed Type I resistance (incidence and severity), have been reported previously. In this study shorter incubation periods, longer latent periods and shorter lesion lengths in the detached leaf assay and higher germination rates in the seed germination assay were related to greater FHB resistance measured by single point inoculation (Type II), collectively explaining 54% of the variation. Overall the relationships observed for Type II FHB resistance were similar to previous findings for Type I resistances. However, the relative magnitude of effects of the individual PDR components determined in vitro varied between FHB disease resistance parameters. Resistance in seed germination and latent period in the detached leaf assay were more strongly related to resistance assessed by point inoculation (Type II) and severity-Type I as opposed to incubation period which was most strongly related to disease incidence-Type I. The results provide evidence that individual components of partial disease resistance differentially affect aspects of FHB disease progression in the wheat spike. This work supports the view that the current model of types of resistance is an oversimplification of the interacting mechanisms underlying expression of FHB resistance.     

A Laboratory Assay for Phytophthora infestans Resistance in Various Solanum Species Reflects the Field Situation

Physiological and molecular research on resistance responses of Solanum tuberosum cultivars and partially resistant Solanum species to Phytophthora infestans requires a reliable resistance test that can be used in the laboratory. Laboratory tests performed on detached leaves and intact plants were compared with field tests for similarity of late blight reactions. Detached leaves from field-grown plants were as resistant as detached leaves from climate chamber-grown plants when challenged with P. infestans. However, detached leaves incubated in covered trays at high relative humidity were more susceptible than detached leaves kept in open trays or leaves on intact plants. The incubation conditions of detached leaves in covered trays rather than detachment itself appeared to affect the resistance expression. Detached leaves of some wild Solanum genotypes became partially infected, whereas intact plants were completely resistant when inoculated. Inoculation of leaves on intact plants, however, resulted in lower infection efficiencies. These limitations should be taken into account when choosing the appropriate inoculation method for specific purposes. For resistance screening, laboratory tests proved to be a good alternative for field tests. The ranking of resistance levels for twenty plant genotypes was similar under laboratory and field conditions.     

Factors affecting the incubation period of dark leaf and pod spot (Alternaria brassicae) on oilseed rape (Brassica napus)

Experiments to investigate the factors affecting the incubation period of dark leaf and pod spot (Alternaria brassicae) on leaves and pods of oilseed rape (Brassica napus) were done in controlled environment (constant temperatures) and glasshouse conditions (fluctuating temperatures). The length of the incubation period of dark leaf and pod spot decreased as infection and incubation temperatures increased from 6 to 20 °C. The incubation period decreased as wetness period increased from 2 to 12 h, as inoculum concentration increased from 80 to 2 × 10³ spores ml^–1 and as leaf age increased from 4 to 10 days. Asymptotes of leaf age and inoculum concentration, above which the length of the incubation period did not decrease, were 10 days and 2 × 10³ spores ml^–1, respectively. The shortest and longest incubation periods were 1 and 11 days. The mechanism by which the infection conditions influenced the incubation period of dark leaf and pod spot on oilseed rape seemed to be linked to lesion density. Usually, the length of the incubation period decreased greatly with increasing lesion density.     

Comparison of screening methods to evaluate the response of St. Augustine grass to Magnaporthe oryzae

The objective of this study was to develop a reliable and high throughput screening method to evaluate the response of St. Augustine grass (Stenotaphrum secundatum) genotypes to the grey leaf spot (GLS) caused by Magnaporthe oryzae infection. Whole plant, detached stolon and detached leaf assays under growth chamber conditions were compared to field conditions on eight commercial and nine advanced breeding lines of St. Augustine grass. Disease was assessed using two variables, lesion size (LS) and overall plant disease severity (SEV). LS and SEV were highly correlated for field and growth chamber screening methods using the whole plant assay (LS r² = 0·79; SEV r² = 0·83; P ≤ 0·001), the detached stolon assay (LS r² = 0·75; SEV r² = 0·72; P ≤ 0·001), and the detached leaf assay (LS r² = 0·46; SEV r² = 0·60; P ≤ 0·001). Genotypic variation for resistance in 17 St. Augustine grass genotypes was identified using all screening methods for LS (P < 0·05) and SEV (P < 0·05). The rank‐sum method was used to classify St. Augustine grass genotypes into highly resistant (HR), resistant (R), moderately resistant (MR), moderately susceptible (MS), susceptible (S) and highly susceptible (HS) classes based on the rank‐sum values of LS and SEV. Two introduced African polyploids used as parents, and two F₁ interploid progeny obtained using an in vitro embryo rescue technique, were classified as highly resistant (HR), or resistant (R), across all screening methods.     

Development of a rapid,accurate glasshouse bioassay for assessing fusarium wilt disease responses in cultivated Gossypium species

A rapid glasshouse‐based bioassay method to screen large numbers of cotton plants for responses to Fusarium oxysporum f. sp. vasinfectum (Fov) was developed. Different Fov inoculum concentrations and methods of inoculation were assessed using resistant and susceptible cotton cultivars. Cotton seeds were planted directly into Fov‐inoculated soil. Studies of seed germination, seedling establishment, seedling mortality and fusarium wilt symptoms (i.e. stunting, foliar symptoms and vascular browning) were performed to optimize the bioassay parameters. Growing seedlings in Fov‐inoculated soils at 5 × 10⁴ or 1 × 10⁵ CFU g^?1 soil, in individual seedling tubes with 12 h at 28–30°C and 12 h at 15–18°C, gave consistent results when assessing Fov disease responses 6 weeks after inoculation. When fusarium wilt resistance ranks (FWRRs) and vascular browning index (VBI) means of 18 Australian and other cotton cultivars from the Fov glasshouse bioassay were compared against their fusarium field performance ranks (F‐ranks), assessed on adult plants for cotton cultivar release, Pearson’s correlation was highly significant for both comparisons. The level of congruence between field and glasshouse data indicated that this protocol should be an effective tool for large‐scale screening for Fov‐resistance responses in diverse germplasm and breeding populations and for advancing genetic research to develop molecular markers for Fov resistance in cotton.     

Resistance and systemic dispersal of Xanthomonas fragariae in strawberry germplasm (Fragaria L.)

The angular leaf spot disease caused by Xanthomonas fragariae is an important plant disease with major impact for the strawberry nursery industry. Currently there is no plant protection product available for controlling the disease effectively. Planting of resistant cultivars seems to be promising, but all commercially used cultivars are susceptible and no donor with a high level of resistance has yet been found. Therefore, a total of 145 genotypes from the Fruit Genebank Dresden (Germany) were evaluated for resistance to X. fragariae by artificial inoculation. Six genotypes were classified as partly resistant, out of which only two (US4808 and US4809) are octoploid. Fragaria vesca f. alba, Fragaria nilgerrensis ‘Yunnan’, F. vesca ‘Illa Martin’ and F. moschata ‘Bauwens’ were also classified as partially resistant, but they are only of limited use for breeding because of their variable ploidy level. Fully resistant genotypes could not be detected. The systemic dispersal of the bacteria in strawberry plants was investigated after inoculation of leaves with X. fragariae strain XF3.9.C and the GFP‐tagged strain XF3.9.C_(pKAN). The systemic spread was evaluated after 3, 7, 14 and 28 days post‐inoculation (dpi) by nested PCR and fluorescence microscopy. After 3 dpi, X. fragariae could be found in all tissues tested including the inoculated leaf, its petiole, the rhizome, the heart bud up to the youngest fully expanded leaf and its petiole. The systemic spread was also detectable in partially resistant genotypes.     

番茄灰叶斑病原菌的鉴定及抗性种质资源的筛选

<正>番茄灰叶斑病是一种世界性病害,近年来,该病在北京、台湾、海南、山东和浙江等地均有发生,极大地限制了我国番茄生产的竞争力~([1])。该病害主要由Stemphylium spp.不同的4个种引起~([2])。本研究采用形态观察法、ITS和gpd序列分析法对从番茄病株分离得到的病原菌进行鉴定,并对番茄灰叶斑病抗性鉴定方法进行研究,以期为番茄灰叶斑病的抗病育种研究及其利用提供理论依据。     

Development ofClonostachys rosea and interactions withBotrytis cinerea in rose leaves and residues

The effect of microclimate variables on development ofClonostachys rosea and biocontrol ofBotrytis cinerea was investigated on rose leaves and crop residues. C.rosea established and sporulated abundantly on inoculated leaflets incubated for 7–35 days at 10°, 20° and 30°C and then placed on paraquat—chloramphenical agar (PCA) for 15 days at 20°C. On leaflets kept at 10°C, the sporulation after incubation on PCA increased from 60% to 93% on samples taken 7 to 21 days after inoculation, but decreased to 45% on material sampled after 35 days. A similar pattern was observed on leaves incubated at either 20° or 30°C. The sporulation ofC. rosea on leaf disks on PCA was not affected when the onset of high humidity occurred 0, 4, 8, 12 or 16 h after inoculation. However, sporulation was reduced to 54–58% on leaflets kept for 20–24 h under dry conditions after inoculation and before being placed on PCA. The fungus sporulated on 68–74% of the surface of leaf disks kept for up to 24 h at high humidity after inoculation, but decreased to 40–51% if the high humidity period before transferral to PCA was prolonged to 36–48 h. The growth ofC. rosea on leaflets was reduced at low inoculum concentrations (10³ and 10⁴ conidia/ml) because of competition with indigenous microorganisms, but at higher concentrations (10⁵ and 10⁶ conidia/ml) the indigenous fungi were inhibited. Regardless of the time of application ofC. rosea in relation toB. cinerea, the pathogen’s sporulation was reduced by more than 99%. The antagonist was able to parasitize hyphae and conidiophores ofB. cinerea in the leaf residues. AsC. rosea exhibited flexibility in association with rose leaves under a wide range of microclimatic conditions, and in reducingB. cinerea sporulation on rose leaves and residues, it can be expected to suppress the pathogen effectively in rose production systems.     

Susceptibility of European pear cultivars to Pseudomonas syringae pv. syringae using immature fruit and detached leaf assays

The susceptibility of thirty-three pear cultivars and two pear rootstocks to four virulent strains of Pseudomonas syringae pv. syringae was evaluated by inoculating detached immature fruits and young leaves. The four strains were similarly virulent and did not show cultivar specificity although they were isolated from different pear cultivars and exhibited different biochemical profiles. The most frequently planted pear cultivars, Conference, Abate Fetel, General Leclerc, Williams, D. Comice, El Dorado, Alexandrine, B. Anjou, Passe Crassane and the rootstock OHxF 333 were susceptible to P. syringae pv. syringae. Maximal severity values were obtained on 'Preguystar' leaves (about 90%). The rootstock Winter Nelis was less susceptible. Results with immature fruit and detached leaf assays agreed with field observations on cultivar susceptibility to bacterial blast. However, the detached leaf test gave a more accurate prediction and has the advantages that symptoms develop quickly (48 h), and leaves are available for a longer period of time than fruits. This method is proposed as a rapid and reproducible screening system of cultivar susceptibility to bacterial blast of pear.     

Towards the Development of a Novel In Vitro Strategy for Early Screening of Fusarium Ear Blight Resistance in Adult Winter Wheat Plants

A novel in vitro bioassay is described for screening Fusarium ear blight (FEB) resistance in adult winter wheat plants. Seven winter wheat cultivars were assessed for components of partial disease resistance as 28 day-old detached leaf segments in the laboratory using isolates of Microdochium nivale var. nivale and M. nivale var. majus. Results were compared with disease data obtained at anthesis using the same cultivars as whole plants and the same isolates under glasshouse conditions. Significant cultivar differences were observed using detached leaves, with cv. Avalon (a Fusarium culmorum ear susceptible cultivar) having the shortest leaf incubation period, greatest leaf lesion development and shortest leaf latent period compared to cv. Spark (a Fusarium culmorum ear resistant cultivar), which had the longest leaf incubation period, least leaf lesion development and longest leaf latent period. Using whole plants, cv. Avalon had the shortest ear incubation period and greatest ear disease severity, whilst cv. Spark had the longest incubation period and least ear disease severity. Overall, cultivars of intermediate F. culmorum ear resistance expressed intermediate responses to M. nivale isolates, using both detached leaves and whole plants. Significant correlations were found with ear disease severity and ear incubation period in whole plants and components of partial disease resistance in detached leaves, with significant correlations obtained between leaf incubation period and ear disease parameters using the M. nivale var. nivale isolate. In addition, leaf latent period and leaf lesion size showed significant correlations with whole plant reactions using M. nivale var. nivale and var. majus isolates. The in vitro screening of cultivars as detached leaves using M. nivale isolates may offer a real possibility of a rapid bioassay for the early screening of FEB resistance in wheat and other cereals.     

Molecular characterization,comparison of screening methods,and evaluation of cross‐pathogenicity of black rot (Xanthomonas campestris pv. campestris) strains from cabbage,choy sum,leafy mustard and pak choi from Taiwan

Choy sum (Brassica rapa var. parachinensis), leafy mustard (Brassica juncea) and pak choi (B. rapa var. chinensis) are highly nutritious components of diets in Taiwan and other Asian countries, and bacterial black rot caused by Xanthomonas campestris pv. campestris (Xcc) is a major biotic constraint in these crops. As very little was known about the Xcc strains from these crops in these regions, including their cross‐pathogenicity and aggressiveness on different hosts, Xcc strains were obtained from cabbage (Brassica oleracea var. capitata), choy sum, leafy mustard and pak choi crops in Taiwan. Two previously published PCR‐based assays reliably distinguished the Xcc strains from other Xanthomonas species and subspecies. Phylogenetic analysis based on repetitive sequence‐based PCR assays placed the Xcc strains in a clade distinct from other Xanthomonas species, and also showed host specificity. Although all of the Xcc strains from the different host species were pathogenic on all five Brassica test species in both a detached leaf assay and an intact plant assay, in the intact plant assay they showed differences in virulence or aggression on the different test hosts. The Xcc strains from leafy mustard and pak choi were consistently highly aggressive on all the test host genotypes, but the strains from choy sum and cabbage were less aggressive on leafy mustard and choy sum. The intact plant assay proved more discriminating and reliable than the detached leaf assay for comparing the aggressiveness of Xcc strains on different host genotypes, and so, with the new Xcc strains isolated in this study, will be useful for screening leafy brassica germplasm accessions for resistance to black rot.     

A comparative assessment of potential components of partial disease resistance to <Emphasis Type="Italic">Fusarium</Emphasis> head blight using a detached leaf assay of wheat,barley and oats

The relative resistance of 15 winter barley, three winter wheat and three winter oat cultivars on the UK recommended list 2003 and two spring wheat cultivars on the Irish 2003 recommended list were evaluated using Microdochium nivale in detached leaf assays to further understand components of partial disease resistance (PDR) and Fusarium head blight (FHB) resistance across cereal species. Barley cultivars showed incubation periods comparable to, and latent periods longer than the most FHB resistant Irish and UK wheat cultivars evaluated. In addition, lesions on barley differed from those on wheat as they were not visibly chlorotic when placed over a light box until sporulation occurred, in contrast to wheat cultivars where chlorosis of the infected area occurred when lesions first developed. The pattern of delayed chlorosis of the infected leaf tissue and longer latent periods indicate that resistances are expressed in barley after the incubation period is observed, and that these temporarily arrest the development of mycelium and sporulation. Incubation periods were longer for oats compared to barley or wheat cultivars. However, oat cultivars differed from both wheat and barley in that mycelial growth was observed before obvious tissue damage was detected under macroscopic examination, indicating tolerance of infection rather than inhibition of pathogen development, and morphology of sporodochia differed, appearing less well developed and being much less abundant. Longer latent periods have previously been related to greater FHB resistance in wheat. The present results suggest the longer latent periods of barley and oat cultivars, than wheat, are likely to play a role in overall FHB resistance if under the same genetic control as PDR components expressed in the head. However the limited range of incubation and latent periods observed within barley and oat cultivars evaluated was in contrast with wheat where incubation and latent periods were shorter and more variable among genotypes. The significance of the various combinations of PDR components detected in the detached leaf assay as components of FHB resistance in each crop requires further investigation, particularly with regard to the apparent tolerance of infection in oats and necrosis in barley, after the incubation period is observed, associated with retardation of mycelial growth and sporulation.     

Bioassays using detached tobacco leaves to determine the sensitivity of Peronospora tabacina to fungicides

Two bioassay methods are described which use detached tobacco leaves to measure the sensitivity of Peronospora tabacina to systemic fungicides. Tobacco leaves (13–15 cm²), treated with fungicides before or after detachment from the plant, were inoculated with sporangia in water drops and, after incubation in beakers and Petri plates, the disease severity and/or production of sporangia was determined 4–7 days after treatment with the fungicides. Of 15 systemic fungicides applied to detached leaves, eight N-phenylamides at 0.066?1.0 μg ml^?1 controlled blue mould; metalaxyl was the most effective fungicide. Isolates of P. tabacina, collected in the field from tobacco plants grown in soil treated with metalaxyl, were not resistant to the fungicide applied to detached leaves prior to inoculation. The fungicide, applied to leaves before detachment, was used to measure the efficacy of five systemic N-phenylamide fungicides sprayed on the basal and unsprayed distal portions of the leaves. Blue mould was controlled on the basal portion of the leaf by all the fungicides at 0.66?1.0 μg ml^?1, but it required the application of 3–30 times more chemical on the basal portion to achieve comparable blue mould control on the distal part of the leaf.     

<Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> occurs asymptomatically on sweet cherry leaves

Leaves of sweet cherry, exposed to either paraquat or freezing to quickly senesce the leaf tissue, were incubated in about 100% RH at 25°C for 6 d. Sporulating colonies of Colletotrichum acutatum, the cause of anthracnose, developed on up to 100% of the paraquat-treated and frozen leaves, and on none of the untreated controls. Number of leaves and leaf area containing C. acutatum on naturally infected leaves increased over time from May to September. Mean incidence of C. acutatum on leaf blades on fruit spurs and vegetative shoots from eight orchard/year samplings were 41 and 33%, respectively. Secondary conidiation (formation of short hyphae and new conidia) from conidia applied to detached leaves took place 6 h after inoculation, but only up to 3% of the conidia formed new conidia. It may be concluded that asymptomatic sweet cherry leaves frequently host C. acutatum and may be a potential inoculum source for cherry fruit.     

A simple detached leaf assay provides rapid and inexpensive determination of pathogenicity of Pythium isolates to ‘all year round’ (AYR) chrysanthemum roots

A detached leaf assay was developed to determine the pathogenicity of Pythium isolates to cut‐flower chrysanthemum roots. Leaves from young plants were excised and inoculated by insertion of a plug of mycelium into a slit cut in the excised petiole. After incubation leaves were assessed for presence and extent of necrosis. Necrosis indicated pathogenicity and was consistently confirmed by comparisons with whole plant inoculations. The rate of necrosis spread also gave some indication of virulence. Isolates of Pythium sylvaticum, P. ultimum and HS group were the most virulent, with a mean rate of spread of 14·6 mm per day, significantly (P < 0·05) faster than the mean rate of spread, 1·6 mm per day, of less virulent isolates. Less virulent isolates included P. irregulare, P. oligandrum and P. aphanidermatum. The latter was unexpected, as P. aphanidermatum is an important species in pythium root rot epidemics in chrysanthemums elsewhere. The value of the detached leaf assay for screening large numbers of isolates was demonstrated in a survey of isolates from clinic samples from chrysanthemum nurseries and in a series of dilution‐plating experiments looking at numbers of Pythium propagules in commercial chrysanthemum beds showing root rot. In the survey, the predominant pathogenic species was identified as P. sylvaticum and the most likely source of infection was contaminated soil as opposed to blocking media or irrigation water, whilst in soil colonization studies the use of detached leaf assays demonstrated a relationship between pathogenic inoculum concentration in soil and the expression of root rot symptoms.     

Prior Inoculation with Non-pathogenic Fungi Induces Systemic Resistance to Powdery Mildew On Cucumber Plants

A spray inoculation of the first leaf of 2-leaf stage cucumber plants with a non-pathogenic isolate of Alternaria cucumarina or Cladosporium fulvum before a challenge inoculation with the pathogen Sphaerotheca fuliginea induced systemic resistance to powdery mildew on leaves 2–5. Systemic resistance was expressed by a significant (p < 0.05) reduction in the number of powdery mildew colonies produced on each leaf of the induced plants, as compared with water-sprayed plants. Systemic resistance was evident when a prior inoculation with each of the inducing fungi was administered 1, 3 or 6 days before the challenge inoculation with S. fuliginea. Increasing the inoculum concentration of A. cucumarina or C. fulvum enhanced the systemic protection and provided up to 71.6% or 80.0% reduction, respectively, in the number of colonies produced on upper leaves, relative to controls. Increasing the inoculum concentration of S. fuliginea used for challenge inoculation, increased the number of powdery mildew colonies produced on both induced and non-induced plants. Pre-treated plants, however, were still better protected than controls, indicating that the level of systemic protection was related to the S. fuliginea inoculum concentration. The induction of systemic resistance against powdery mildew by biotic agents, facilitates the development of a wide range of disease management tools.