Induced resistance: a tool for fungicide resistance management

Induced resistance to the apple scab fungus Venturia inaequalis was demonstrated in greenhouse tests with 12-day-old seedlings of the apple cultivar Golden Delicious treated with methyl 2,6-dichloro-isonicotinate or 3,5-dichlorosalicylic acid prior to inoculation with the causal fungus. Studies of the dose-response of flusilazole on induced resistant plants revealed synergistic effects between both crop protection principles. Therefore, the use of such resistance-inducing compounds in the field might allow a reduction in the number of fungicide applications, and possibly a reduction in dose, thus resulting in improved efficacy of fungicides. There was also evidence that induced resistance could prove to be a valid strategy for the treatment of pathogen populations with reduced sensitivity to a given fungicide. © 1998 SCI     

CCK1, a PMK1-type MAP kinase is required for hyphal growth,pigmentation, conidiation,enzyme activity,osmotic stress response,and pathogenicity in <Emphasis Type="Italic">Corynespora cassiicola</Emphasis>

The aim of this study was to confirm the presence of races in populations of the fungus Venturia inaequalis that are able to overcome specific apple scab resistance gene(s) within the major apple-growing areas of Poland. The monitoring was conducted in six orchards located in the north, centre and south of Poland. The study involved the use of 16 differential genotypes for pathogenicity testing conducted under both greenhouse and orchard conditions. In addition, the occurrence of apple scab on 10 apple cultivars containing the Rvi6 gene was assessed in four organic orchards in central Poland. Apple scab was found on their leaves for the first time in Poland in 2010. The use of differential genotypes containing specific resistance genes suggested that 10 apple scab resistance genes had been overcome by V. inaequalis in the orchards monitored in this study.     

Characterization of the defence response to Venturia inaequalis in ‘Honeycrisp’ apple,its ancestors,and progeny

The apple cultivar Honeycrisp exhibits genetic resistance to apple scab. The characterization of the macroscopic and microscopic responses on leaves infected by the pathogen Venturia inaequalis is described. The macroscopic resistance reactions observed in ‘Honeycrisp’, its parent ‘Keepsake’, and grandparents ‘Frostbite’ and ‘Northern Spy’ ranged from 0 (no reaction) to chlorotic flecking, stellate chlorosis, necrotic flecking, and sporulation. No hypersensitive response was observed. The resistance response occurred as early as 7 days post inoculation (dpi) at the same time that susceptible plants exhibited macroscopic signs of the disease. The resistance reactions were similar in the progeny population of ‘Honeycrisp’ × ‘Twin Bee Gala’, although they were delayed to 10–14 dpi possibly due to variable greenhouse conditions. This population segregated 3 resistant:1 susceptible, which suggests the presence of two genes in ‘Honeycrisp’ and agrees with the finding that different responses within ‘Honeycrisp’ to mixed inoculum are due to differential recognition of pathogen effectors.     

A new scab‐like disease on apple caused by the formerly saprotrophic fungus Venturia asperata

Atypical scab‐like symptoms were reported for the first time in 2007 in the south of France on fruits of apple cultivars carrying the Rvi6 (=Vf) major resistance gene to Venturia inaequalis. With microscopic observations, nucleotide sequence data and pathological tests, it was shown that the causal agent was Venturia asperata. Scanning electron microscopy was used to compare its infection process and conidiogenesis to those of Venturia inaequalis on apple and Venturia pirina on pear. Venturia asperata produced fewer hyphae and fewer spores than the two other Venturia species, and resulted in weaker symptoms. This fungal species was previously described as a saprotroph on apple leaf litter. This is the first report of damage on apple fruits caused by V. asperata. Changes in host and cultural practices may have created a new context favourable for the emergence of this pathogen. It was also detected on symptomless leaves and on overwintered leaves on the ground. Pseudothecia developed on overwintered leaves and released ascospores over a 2‐month period from the end of March until the end of May, suggesting that the fungus is able to survive from season to season. However, it is not yet known if this new disease will establish over coming years and become an emergent disease.     

A rapid virulence assay for the Dutch elm disease fungus Ophiostoma novo‐ulmi by inoculation of apple (Malus × domestica ‘Golden Delicious’) fruits

Large‐scale virulence tests using trees or saplings are expensive, time‐consuming and require a considerable amount of space. The suitability of using ‘Golden Delicious’ apples as a rapid screen for identifying Ophiostoma novo‐ulmi transformants with reduced virulence was thus evaluated. When a collection of O. novo‐ulmi field isolates belonging to subspecies novo‐ulmi or americana was inoculated to apples, members of subsp. novo‐ulmi induced, on average, larger necrotic lesions than subsp. americana isolates. The size of the lesions on apples was not correlated with mycelial growth rate of isolates on nutrient agar. Insertional mutants from O. novo‐ulmi subsp. novo‐ulmi isolate H327 were inoculated to ‘Golden Delicious’ apples and Ulmus parvifolia × U. americana saplings in parallel experiments. Results clearly indicated that the O. novo‐ulmi transformants included several exhibiting significantly altered levels of virulence. Variability among replicates within a treatment was reduced in apple inoculation data compared to elm sapling data. Overall, the ‘Golden Delicious’ apple assay was found to be an excellent means for rapidly assessing the virulence level of O. novo‐ulmi isolates.     

A method to monitor airborne Venturia inaequalis ascospores using volumetric spore traps and quantitative PCR

Apple scab caused by the fungus Venturia inaequalis can result in significant crop losses if not managed effectively. Sanitation as part of an integrated management strategy aims to significantly reduce primary inoculum to lower the disease pressure. This study evaluates the possibility of molecular detection and quantification of ascospore discharge and the use of this method to test for efficacy of orchard sanitation treatments. A method to detect and quantify airborne ascospores was developed using volumetric spore traps (VSTs). V. inaequalis specific primers were tested on daily VST samples from two orchard sections (leaf litter removed compared to leaf litter left) during spring. A molecular method to detect and quantify ascospores was tested by amplifying genomic regions of the mitochondrial CYP51A1 gene, and the ITS region using SYBR® green. Timing of ascospore discharge was compared to predicted infection risk and a degree day model using weather data. The average spore detection limit was estimated to be at levels of 1 pg μl^?1 DNA (approximately 37 ascospores) per daily spore trap reading using CYP51A1 primers. Using the CYP51A1 primer pair, primary inoculum was estimated to be 51 % lower in the orchard sections where leaves had been removed, indicating that this method could be used to evaluate the efficacy of alternative control strategies such as leaf removal to reduce potential ascospore dose. This is the first report of combining VSTs and quantitative PCR to monitor airborne V. inaequalis ascospores.     

Within‐ and between‐orchard variability in the sensitivity of Venturia inaequalis to myclobutanil,a DMI fungicide,in the UK

BACKGROUND: Myclobutanil, a demethylation inhibitor (DMI) fungicide, is an important fungicide for controlling apple scab and powdery mildew. Overuse of this fungicide has led to establishment of scab isolates with reduced sensitivity to this fungicide in several countries. Experiments were conducted to determine the sensitivity of the causal agent of apple scab, Venturia inaequalis (Cooke) Winter, to myclobutanil in the UK, in order to assess whether there is a relationship between fungal insensitivity and the number of DMI applications, and establishing whether fungal sensitivity varied greatly within an orchard. RESULTS: Reduced sensitivity of V. inaequalis to myclobutanil was positively related linearly to the number of DMI applications. ED₅₀ values ranged from 0.028 to 1.017 mg L^?1 (average = 0.292) for the baseline population, whereas isolates from two other orchards had much greater ED₅₀ values, ranging from 0.085 to 5.213 mg L^?1 (average = 1.852). There was significant variation in fungal sensitivity to myclobutanil among fungal isolates from different locations within a single orchard. CONCLUSIONS: Spatial spread of insensitive isolates of V. inaequalis to myclobutanil is likely to be limited in distance. Conidia may be an important source of primary inoculum. Myclobutanil should still be effective for most field isolates, but its use should be strategically integrated with other groups of fungicides. Copyright © 2009 Society of Chemical Industry     

Concepts and methods in biological control of diseases in apple orchards1

The project ‘Promotion of Biological Methods in Apple Growing’ which started in Switzerland in 1985 concentrates on the control of Venturia inaequalis (scab) and Podosphaera leucotricha (powdery mildew). The project considers the selection of suitable cultivars resistant or only slightly susceptible to fungal diseases and the assessment of tolerable economic loss. It also includes curative treatments with sterol-synthesis-inhibiting fungicides, according to scab infection periods, as well as applications of plant extracts (proposed by the Institute of Biological Husbandry) and suspensions of antagonists or their culture filtrates as plant-protecting agents. Screening experiments on apple seedlings showed that root extracts of Rumex obtusifolium significantly reduced powdery mildew infections under greenhouse conditions. In field experiments treatments with R. obtusifolium extracts at intervals of 7–10 days were less effective. Saponin-containing extracts of different plants gave satisfying control of scab on seedlings in the growth chamber but were not very effective in the field. Applications of spore suspensions and culture filtrates of the phyllosphere fungi Chaetomium spp. reduced scab and mildew infections on apple seedlings to some extent. It was found that in the last 2 years even primary infections of P. leucotricha were severely parasitized by Ampelomyces quisqualis. Preliminary studies showed that the lack of efficacy of this biocontrol system is due to the delayed spread of the hyperparasite.     

Comparative studies on the effects of a yucca extract and acibenzolar-<Emphasis Type="Italic">S</Emphasis>-methyl (ASM) on inhibition of <Emphasis Type="Italic">Venturia inaequalis</Emphasis> in apple leaves

The effect of an extract of Yucca schidigera on the control and infection process of the apple scab pathogen, Venturia inaequalis, was examined and compared with the chemical resistance inducer, acibenzolar-S-methyl (ASM). In seedling assays, both materials significantly reduced apple scab symptoms and pathogen sporulation on leaves and both showed similar control efficacies as the reference treatment, sulphur. Whereas yucca extract and sulphur gave significant inhibition of conidial germination in vitro, ASM did not inhibit germination. Histopathological studies of the infection process of V. inaequalis in apple leaves showed that the yucca extract primarily acted by inhibiting pre-penetration events and penetration itself. In contrast, the ASM treatment significantly inhibited more stages of the infection process (pre-penetration, penetration and post-penetration events). These observations suggest that the yucca extract acted mainly by a direct fungitoxic effect whereas ASM, as expected, acted as a resistance inducer. However, expression studies of two genes encoding the PR proteins, PR1 and PR8, in apple seedlings indicated that yucca extract may also affect plant defence as expression of both genes was up-regulated following yucca treatment, to a level similar to that observed after treatment with ASM. The fungitoxic effect of sulphur on V. inaequalis was also confirmed in this study.     

In vitro screening for resistance to apple proliferation in Malus species

A screening system for apple proliferation resistance was developed, based on in vitro graft‐inoculation with the causal agent ‘Candidatus Phytoplasma mali’. For this, in vitro cultures of the field‐resistant apomictic genotypes Malus sieboldii, H0909, D2212 and the susceptible Malus × domestica genotypes Golden Delicious and rootstock M9 were established, as well as in vitro cultures of Rubinette and Golden Delicious infected with ‘Ca. P. mali’ strains PM4 and PM6, respectively. Healthy in vitro shoots were inoculated by micrografting with infected shoots used as graft tip. After 6 weeks graft contact no significant differences for graft quality were observed between healthy and infected grafts. Mortality of grafts and transmission rates were not significantly different among the different genotypes. The phytoplasma concentration in inoculated shoots was determined at different times post‐inoculation (p.i.) by quantitative real‐time PCR. Infected M. sieboldii and D2212 had lower phytoplasma concentration than the susceptible controls and showed no symptoms. H0909 showed an intermediate behaviour exhibiting lower phytoplasma concentrations with strain PM4 but growth was affected. The dynamics of phytoplasma concentration reached a maximum at 6–8 months p.i. for all genotypes but the values for 3–5 and 10–12 months p.i. were similar. The resistance of M. sieboldii and D2212 was confirmed in vitro. A significant difference in phytoplasma concentration was observed between strains PM4 and PM6.     

Differentiation in populations of the apple scab fungus Venturia inaequalis on cultivars in a mixed orchard remain over time

The ascomycete Venturia inaequalis causes annual epidemics of apple scab worldwide. Scab development is reduced in mixed cultivar orchards compared with monocultures. In order to use mixtures in commercial production, how the population of scab changes in a mixed orchard needs to be understood, together with how likely a super race, with virulence factors overcoming multiple resistance factors in the mixed orchard, is to emerge and become dominant. This study used simple sequence repeat (SSR) markers to investigate the temporal change of scab populations in two mixed cultivar orchards in the UK to infer the likelihood of emergence of a scab super race. There were no significant differences between the populations at the two sampling times (6 or 7 years apart) in either of the two mixed orchards. In one of the orchards, apple scab populations on different cultivars were significantly different and the differences did not diminish over time. These results suggest that it is not inevitable that a super race of V. inaequalis will become dominant during the lifetime of a commercial apple orchard.     

Characterisation of a protein from Venturia inaequalis that induces necrosis in Malus carrying the Vm resistance gene

Apple scab (black spot) is caused by the fungus, Venturia inaequalis. Race 1 isolates of this fungus are avirulent on Malus hosts carrying the resistance gene V_m. Detached leaves from a V_m host (resistant, differential host 5) and ‘Royal Gala’ (susceptible, host 1) were inoculated with a conidial suspension of V. inaequalis. In the resistant reaction, a hypersensitive response (HR), characterised by necrosis and the accumulation of autofluorescent materials in epidermal and mesophyll cells, was observed at the site of fungal penetration. No HR was observed in the susceptible host. V. inaequalis grown in vitro produced an elicitor that induced necrosis, similar to the HR, when infiltrated into leaves of the resistant V_m host. No response, however, was observed in the susceptible host. The elicitor was proteinaceous and a fraction with elicitor activity was isolated using ultra-filtration, acetone precipitation and ion-exchange chromatography. The elicitor activity was resistant to boiling but it was abolished by digestion with proteinase K. The protein fraction contained three major proteins all with low isoelectric points (pI 3·0–4·5). The fraction also elicited necrosis in the differential host 4, but not in any of the other resistant hosts tested, including differential hosts 2, 3, and 6. Therefore, the fraction may contain elicitors with more than one host specificity.     

The threat of wild habitat to scab resistant apple cultivars

Evaluations of plant resistance to pathogens are rarely made using isolates from wild habitats, although the heterogeneity of such habitats may generate pathogen diversity which could be a source of new virulence in cultivated habitats. The aim of this study was to investigate whether scab resistance factors, identified and characterized in apples using isolates of Venturia inaequalis from a cultivated habitat, remained effective against isolates from a wild habitat. Three V. inaequalis core collections originating from the cultivated apple Malus × domestica and from two wild species, M. sieversii and M. sylvestris, were established to maximize pathogen diversity. For each core collection, 10 isolates were inoculated in mixtures onto 51 genotypes from an apple progeny segregating for two qualitative resistance genes and six quantitative resistance loci (QRL). On each apple genotype, isolates that contributed to the scab symptoms were identified within the mixture using microsatellite markers. The most frequently detected isolates were inoculated singly to compare their aggressiveness according to their host origin. The results showed that isolates from a wild habitat were able to infect the susceptible apple genotypes. However, these isolates were never more aggressive than isolates from the cultivated habitat on the resistance factors tested. It can therefore be concluded that the resistance factors used in this study, identified with V. inaequalis isolates from a cultivated habitat, remained effective against isolates from M. sylvestris and M. sieversii.     

Evaluation of a pheromone trap for the cotton pink bollworm Pectinophora gossypiella Saunders

This 4-year study examined the susceptibility of nine cultivars of apple, Malus domestica Borkhausen to attack by woolly apple aphid (WAA), Eriosoma lanigerum in Jordan. The cultivars under investigation were: Prima Rouge, Delberd Steval, Early Gold, Starking Delicious, Harmony, Golden Smoothee, Fuji, Melrose, and Golden Delicious. Estimation of susceptibility was based upon a numeric scale of 0–4 for both root and shoot, but each with different specifications. A tree infestation rating was obtained by calculating the average rating of both edaphic and arboreal ratings of each tree. Fuji was significantly the most infested cultivar during the period of study and it ranked as a highly susceptible cultivar. Prima Rouge, Early Gold, Starking Delicious, Golden Smoothee, and Golden Delicious showed lower susceptibility. Harmony was an immune cultivar as it sustained neither edaphic (root inhabiting) nor arboreal colonies of woolly apple aphid. We propose the development of rootstocks from Harmony cultivar. The latter offers a new gene for resistance to WAA that will open the door for plant breeders to produce different resistant rootstocks.     

Using real-time PCR to survey frequency of azoxystrobin-resistant allele G143A in Alternaria populations from almond and pistachio orchards in California

Alternaria spp. cause leaf spot of almond and Alternaria late blight of pistachio in California, and azoxystrobin is a strobilurin fungicide that has been registered for the control of these diseases. To date, only a single point mutation of G143A in cytochrome b resulting to azoxystrobin resistance in Alternaria spp. was found in California. Based on this single point mutation, a real-time PCR assay was developed to quantify the frequency of the resistant allele G143A (FA) in pathogen samples taken from orchards. Forty-one almond and pistachio orchards were arbitrarily selected in eight counties of California. Fifty leaf lesions caused by Alternaria spp. per orchard were cut to extract the fungal DNA for a real-time PCR assay to determine the FA. About 88% of 41 surveyed orchards had Alternaria spp. with FA > 0.90, while six pistachio orchards showed a FA < 0.90. Therefore, azoxystrobin-resistant Alternaria populations are predominant in almond and pistachio orchards in California, and sprays of azoxystrobin to control Alternaria diseases are not recommended in these orchards. This study shows a potential use of a real-time PCR assay to efficiently quantify the frequency of azoxystrobin-resistant Alternaria spp. from large number of samples.     

Selection and orchard testing of antagonists suppressing conidial production by the apple scab pathogen <Emphasis Type="Italic">Venturia inaequalis</Emphasis>

Apple scab caused by Venturia inaequalis is a major disease in apple production. Epidemics in spring are initiated by ascospores produced on overwintering leaves whereas epidemics during summer are driven by conidia produced on apple leaves by biotrophic mycelium. Fungal colonisers of sporulating colonies of V. inaequalis were isolated and their potential to reduce the production of conidia of V. inaequalis was evaluated on apple seedlings under controlled conditions. The four most effective isolates of the 63 screened isolates were tested subsequently under Dutch orchard conditions in 2006. Repeated applications of conidial suspensions of Cladosporium cladosporioides H39 resulted in an average reduction of conidial production by V. inaequalis of approximately 40%. In 2007, applications of conidial suspensions of C. cladosporioides H39 reduced conidial production by V. inaequalis by 69% on August 6 and by 51% on August 16, but no effect was found on August 20. However, viability of available conidia of C. cladosporioides H39 was low at the end of the experiment. Epiphytic and endophytic colonisation by Cladosporium spp. of leaves treated during the experiment with C. cladosporioides H39 was significantly higher than on control leaves sampled 6 weeks after the last application. It is concluded that C. cladosporioides H39 has promising potential as a biological control agent for apple scab control. More information is needed on the effect of C. cladosporioides H39 on apple scab epidemics as well as on mass production, formulation and shelf life of conidia of the antagonist.     

Detection and quantification of Rhizoctonia cerealis in soil using real-time PCR

Rhizoctonia cerealis E.P. van der Hoeven (anamorph of Ceratobasidium cereale D.I. Murray and Burpee), which causes sharp eyespot in wheat, is a major soilborne fungal pathogen that severely impairs yield and quality of winter wheat in China. Because the pathogen is difficult to identify and quantify in soil using conventional methods, a rapid and reliable method is needed to detect and quantify the fungus. In this study, we developed an SYBR Green-based quantitative real-time polymerase chain reaction assay for specific, sensitive detection and quantification of R. cerealis in soil samples. Using a specific primer pair based on the β-tubulin gene of the fungal DNA sequence, we could specifically detect R. cerealis at quantities as low as 100?fg of purified pathogen DNA. Using the real-time PCR assays, we were able to quantify R. cerealis in artificially and naturally infested soil samples. This new technique to quantify R. cerealis is rapid and accurate and will be a useful tool for future studies of pathogenic R. cerealis.     

Metabolism of amitrole in apple: II. Bound residues from mature fruits

Significant radioactivity detected in mature fruits, harvested from apple trees (Malus domestica Borkh., cv. ‘Golden Delicious’ and ‘Gloster’) that were soiltreated with [3,5-¹⁴C]amitrole, remained in the insoluble plant material after exhaustive extraction. These bound residues were solubilized with a mixture of pectinases and cellulases. Thus, separation and characterization of carbohydrates and xenobiotic moieties released during this procedure became possible. A part of the radiolabel was incorporated into natural products, indicating degradation of the applied amitrole and reassimilation of [¹⁴C] carbon dioxide.     

Population variation of apple scab (Venturia inaequalis) within mixed orchards in the UK

Apple scab, caused by Venturia inaequalis, is one of the most important apple diseases worldwide. To investigate between- and within-orchard fungal variability, 212 isolates were sampled from two mixed orchards, one of 10?years of age and the other of 45?years of age, in the UK and genotyped with AFLP and SSR markers. Populations of isolates from the two orchards did not differ significantly in terms of allele frequencies at the screened AFLP and SSR loci. However, groups of isolates from individual cultivars differed significantly within each orchard and there were also significant differences between groups of isolates from individual trees of the same cultivar in the same orchard. These differences were less pronounced in the younger mixed orchard than in the older one. The existence of tree-to-tree fungal variability indicates a possible role for conidia as a source of primary inoculum. Non-random mating may be one of the factors causing the significant differences among fungal populations from different cultivars. These results suggest that apparently ??susceptible?? cultivars have different background genetic resistance factors, which can be exploited for disease management in mixtures.