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.     

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.     

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.     

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.     

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.     

A during-infection spray strategy using sulphur compounds,copper, silicon and a new formulation of potassium bicarbonate for primary scab control in organic apple production

In a field experiment conducted over two growing seasons, the effectiveness and phytotoxicity of inorganic fungicides such as sulphur, lime sulphur, copper, silicon and Armicarb (a new formulation of potassium bicarbonate) was compared with water for the control of primary apple scab infections in Belgium on high, medium and low scab-susceptible cultivars (cvs ‘Pinova’, ‘Pirouette’ and ‘Reinette des Capucins’, respectively). In order to drastically reduce the amount of fungicide applied in the orchard, two approaches were used: (1) a strategy involving spraying during the infection process, before fungal penetration and (2) a tunnel sprayer machine for treatment applications. Under field conditions highly favourable for disease, low rates of elemental sulphur (31.8 and 38.6 kg ha⁻¹ year⁻¹ in 2005 and 2006, respectively) combined with low rates of copper (2.1 kg ha⁻¹ year⁻¹ in both years) provided the best scab control and reduced scab severity on the fruits of cv. ‘Pinova’ by 97 and 98% compared with water control in 2005 and 2006, respectively. Lime sulphur was much more effective than wettable sulphur and appeared to be efficient at temperatures below 10°C, but its effectiveness against apple scab decreased if the treatments were applied 12–24 h later than in the ‘during-infection’ spray strategy. Armicarb used alone significantly reduced apple scab severity on the leaves and fruits of the three cultivars compared with the water control. Its effectiveness was as good as wettable sulphur applied using the same timing and dosage. Silicon reduced apple scab on fruits very slightly, but not on leaves. The amounts of wettable sulphur, lime sulphur, copper, silicon and potassium bicarbonate used in this experiment to control apple scab were not phytotoxic, did not increase fruit russet, did increase the yield of each cultivar and did not affect summer density of the beneficial Typhlodromus pyri. The potential and limitations of ‘during-infection’ spraying as a protection strategy against apple scab in organic farming are discussed.     

Evidence for loss of ontogenetic resistance of apple leaves againstVenturia inaequalis

The susceptibility of apple leaves toVenturia inaequalis was investigated by assessing disease on individual leaves of seedlings and labelled shoots of orchard trees. Four sets (A-D) of potted seedlings of cv. Golden Delicious, which had been grown in a glasshouse, each with approximately 30 mature leaves, were exposed to a high-inoculum orchard. Sets A and B were exposed after each other for 47 and 42 days, respectively. As a result of the six and three infection periods during exposure, 94% and 81%, respectively of the seedling shoot tips in set A and set B became infected. However, due to ontogenetic resistance, disease incidence was low in both sets on leaves which were fully expanded at the beginning of exposure. Set C was exposed during both periods (89 days) in which sets A and B were exposed. Not only were all the seedling shoot tips in set C infected, but also — due to the loss of ontogenetic resistance — nearly all of the mature leaves. Ontogenetic resistance was also lost in set D, which was exposed for 57 days at the end of the growing season. The symptoms on fully expanded leaves on plants in sets C and D were typical and similar to those on young leaves. A time course symptom assessment was performed on leaves which had developed early in the season on labelled, field-grown shoots of cv. Golden Delicious trees. A steady increase of disease incidence was detected, which could not only have resulted from infections followed by extended incubation periods. In addition to the increase of typical lesions on both leaf surfaces, there was also an abundance of diffuse mycelial development on the lower surfaces of the leaves of the field-grown trees.     

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.     

Accumulation of phenolic compounds in apple in response to infection by the scab pathogen,Venturia inaequalis

The research dealt with phenolics in healthy versus scab infected apple leaves and fruits. The leaf samples were picked in the period from May to September and the fruit samples at technological maturity. Infection with the Venturia inaequalis fungus enhanced the metabolism of phenolics at the infected sites, especially in the boundary tissue. Tissue infected with Venturia inaequalis showed in comparison to the healthy tissue up to 7.6 times more hydroxycinnamic acids, up to 2.6 times more flavan-3-ols and up to 2.9 times higher values of flavanols. The content level of total phenolics in the infected tissue was 1.3–2.4 times higher than in the healthy leaves and fruit.     

Can phyllosphere yeasts explain the effect of scab fungicides on russeting of Elstar apples?

In 1999 and 2000, the effects of scab fungicides on yeast composition and russeting of Elstar apples were assessed. Yeast composition of fungicide-treated and untreated young apple fruit with or without russet symptoms was investigated and enzyme activity of the yeasts was studied. Cryptococcus albidus, C.laurentii,Rhodotorula glutinis, Sporobolomyces roseus andMetschnikowia pulcherrima dominated the apple fruit surface. Russeted apple fruitlets had a higher red yeast density than non-russeted fruitlets. Invitro fungicide susceptibility of the dominant yeast species varied. Dithianon and dodine were active against all tested species, captan and tolylfluanid showed specificity for certain species, whereas pyrimethanil and bupirimate were largely ineffective. Fungicide treatment in the field had a clear effect on the yeast composition. Metschnikowia etschnikowia pulcherrima was eliminated from the phyllosphere and cryptococco$ï species diminished by both captan and dithianon. The red yeast population was not significantly changed by either fungicide. Captan, dithianon, tolylfluanid and pyrimethanil reduced russet in the field in both years, dodine and kresoxim-methyl only in 2000. All yeast species had cutinolytic activity, all but M.pulcherrimaand Debaryomyces hansenii were lipolytic, and some of the isolates showed proteolytic activity.     

Molecular Identification of a Sexual Interloper: The Pear Pathogen, Venturia pirina, has Sex on Apple

ABSTRACT Venturia pirina (the pear scab pathogen) and V. inaequalis (the apple scab pathogen) were detected as ascospores discharged from apple leaf litter in New Zealand (spring 1998). Pseudothecia of both species were located on dead apple leaves; however, only those of V. inaequalis were associated with scab lesions. V. pirina was identified by rDNA sequence analyses, because morphological characters could not distinguish this fungus from V. asperata (a rare saprophyte on apple) and other Venturia spp. pathogenic on rosaceous fruit trees. Species-specific polymerase chain reaction primers designed to the 18S end of the internal transcribed spacer 1 region differentiated Venturia fruit tree pathogens reliably. V. pirina field isolates were pathogenic on pear, but only weak saprophytes on apple. In rare instances, when appressoria of V. pirina appeared to penetrate the cuticle of apple leaves, epidermal cells responded with a localized hypersensitive response (HR). To our knowledge, this is the first report of induction of HR-like events by V. pirina on its nonhost, apple, and also the first record of sexual reproduction of V. pirina on apple. It is assumed that V. pirina pseudothecia formed from saprophytic lesions in senescing apple leaves when active defense mechanisms such as HR were no longer induced.     

Real-time PCR as a promising tool to monitor growth of Venturia spp. in scab-susceptible and -resistant apple leaves

Apple scab, the most important disease of apple worldwide, is caused by Venturia inaequalis. Currently, evaluation of fungal pathogenicity and host resistance is based on a symptomatic disease rating. However, this method does not provide an accurate measurement of the degree of infection and cannot detect early fungal development in symptomless leaves. In this study, a Venturia-specific real-time PCR assay was developed using primers designed around the specific internal transcribed spacer 2 (ITS2) region of the 5.8S rRNA gene. Using SYBR? Green I technology, the assay can accurately quantify Venturia DNA over a concentration range of at least five orders of magnitude. Detection sensitivities were in the order of 100?fg. The method was used to quantify Venturia genomic DNA levels in leaves of three apple cultivars with different levels and types of scab resistance and artificially infected with V. inaequalis. The assay clearly discriminated between Venturia levels in monogenic resistant (‘Topaz’), polygenic resistant (‘Discovery’), and susceptible (‘Golden Delicious’) cultivars, and proved especially useful to quantify pathogen levels during the initial latent stage of infection. The real-time PCR data of ‘Golden Delicious’ were consistent with the observed evolution of the degree of sporulation during a time-course experiment. Although measurements were influenced by the presence of co-extracted PCR-inhibitors, the impact of these compounds was independent of the apple cultivar or the initial amount of fungal DNA present. In conclusion, real-time PCR amplification of the ITS2-5.8S rDNA of Venturia spp. is a faster, more objective and more sensitive method to monitor fungal growth and to evaluate host resistance than phenotypic disease rating scores.     

Genomic sequencing indicates non-random mating of Venturia inaequalis in a mixed cultivar orchard

Apple scab is one of the most economically important diseases of apples worldwide. The disease is caused by the haploid ascomycete Venturia inaequalis. Growing apples in cultivar mixtures may reduce disease severity. To determine how the pathogen population structure is affected by host mixtures we studied 24 V. inaequalis isolates sampled from three different apple cultivars (Bramley, Cox, and Worcester) growing in a mixed orchard approximately 50 years old. The isolates were aligned against a reference genome and single nucleotide polymorphisms (SNPs) were called between the isolates. The populations isolated from Bramley and Worcester were distinct, while Cox isolates were an admixture. This supports previous tests of the ability of isolates to cross-infect hosts, and molecular comparisons using simple sequence repeats (SSRs). Genotype-specific allele (GSA) loci were not distributed randomly across contigs in proportion to contig length, but were clustered. Clustered GSA loci were observed in almost all contigs. This indicates population differentiation across the whole genome, presumably due to lack of crossing-over events between Bramley and Worcester isolates. This lack is probably due to physical separation effects: sexual mating is more likely to take place and succeed between isolates from lesions on the same leaf than from contact between independently infected leaves in leaf litter on the orchard floor. This would especially be the case if sexual reproduction is initiated before leaf-fall.     

苹果黑星病在辽宁省的分布及其发生规律

苹果黑星病是辽宁省植物检疫对象。目前主要分布于小苹果栽植区及大、小苹果混栽区,已接近辽宁南部的大苹果主产区。经自然感病和人工接种证明,大、小苹果上的黑星病菌可以交互侵染,目前一些主栽的大苹果品种均能感染此病。辽宁地区苹果黑星病的初次侵染来源是落地越冬病叶上于翌春产生的子囊孢子。分生孢子不能越冬成活。苹果枝条及芽鳞不带菌。黑星病菌的两种孢子随气流传播,使病害逐步扩大蔓延。经过6年的观察,苹果黑星病发生始期早晚及发病轻重,与早春和夏季的降水量多少成正相关。田间药剂防治试验结果,以托布津、乙磷铝、多菌灵、特克多等药剂防治效果良好。     

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.     

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     

Incidence of Verticillium wilt of artichoke in eastern Spain and role of inoculum sources on crop infection

Surveys of 94 artichoke fields throughout the artichoke production areas of Comunidad Valenciana (eastern Spain) were conducted from 1999 to 2002 to determine the incidence and distribution of Verticillium wilt.Verticillium dahliae was isolated from 80.9% of the sampled fields, and detected in all artichoke-growing areas, with a mean disease incidence of 53.8% infected plants. The disease was found to cause severe damage to cv. ‘Blanca de Tudela’, which is the most important artichoke cultivar grown in Spain, and was also observed on the seed-propagated cv. ‘Imperial Star’. In field trials to study the role of infected planting material and soil inoculum on infection of artichoke plants during the cropping season,V. dahliae was transmitted from infected stumps to the plants, confirming that the use of infected stumps could have greatly contributed to the dissemination of the pathogen. Inoculum density ofV. dahliae in soil had an effect on crop infection, in that a higher number of microsclerotia per gram of soil resulted in a higher percentage of infected plants. In addition, yield of cv. Blanca de Tudela was significantly affected byV. dahliae infection, showing that a higher percentage of infection corresponded with lower yield. http://www.phytoparasitica.org posting July 21, 2005.     

Polygalacturonase-inhibiting protein (PGIP) from Japanese pear: possible involvement in resistance against scab

Venturia nashicola is the causal agent of scab, a fungal disease affecting Asian pears. The Japanese pear cv. ‘Kousui’ is highly susceptible to the race 1 of this fungus whereas the cv. ‘Kinchaku’ and the non-host European pear cv. ‘Flemish Beauty’ are resistant. The aim of this work is to investigate the role of polygalacturonase-inhibiting proteins (PGIPs) of pear during the interactions with V. nashicola leading to susceptibility or resistance. PGIP protein was detected from immature fruit of Kousui and Kinchaku. It showed a molecular mass of 42 kDa that shifted to 35 kDa after chemical deglycosylation. The gene pgip was amplified by PCR using genomic DNA and/or cDNA from young leaves of Kousui, Kinchaku, and European pear cvs. Flemish Beauty, ‘Bartlett’, and an Asian wild pear strain ‘Mamenashi 12’, then sequenced after sub-cloning. Some conserved variations were identified in the sequence indicating that gene family also exists in pgip of Japanese pear and confirmed by Southern blot analysis. The expression of PGIP was studied in scab-inoculated leaves of the susceptible Kousui and the resistant Kinchaku and Flemish Beauty. pgip Gene and its encoding protein were highly and rapidly activated in these resistant plants. In addition, PGIP extracts derived from Kinchaku and Flemish Beauty partially inhibited the activity of polygalacturonase (PG) from V. nashicola suggesting a possible role of PGIP in limiting fungal growth frequently observed in these resistant cultivars.