Identification of Neofabraea species causing bull's eye rot of apple in Poland and their direct detection in apple fruit using multiplex PCR

Based on partial sequence analysis of the β‐tubulin gene, 19 isolates of fungi causing bull's eye rot on apple in Poland were classified into species: Neofabraea alba, N. perennans and N. kienholzii. To the authors’ knowledge, the detection of N. kienholzii is the second in Europe and the first in Poland. Species affiliation of these fungi was confirmed by a new species‐specific multiplex PCR assay developed on the basis of previously published methods. The new protocol allowed for the specific identification of bull's eye rot‐causing species, both from pure cultures and directly from the skin of diseased or apparently healthy apples. In 550 samples of diseased fruits collected from nine cold storage rooms located in three regions of Poland, in 2011 and 2012, N. alba was detected as the predominant species causing bull's eye rot, occurring on average in 94% of the tested samples. Neofabraea perennans was found in a minority of apple samples, N. kienholzii was found only in two apple samples, while N. malicorticis was not detected in any sample tested. In tests on 120 apparently healthy fruits, only N. perennans was detected in a single sample. The results of genetic diversity analyses of bull's eye rot‐causing fungi based on the β‐tubulin gene sequence and an ISSR (inter‐simple sequence repeat) PCR assay with two primers were consistent, showing the expected segregation of tested isolates with respect to their species boundaries. However, the genetic distance between N. perennans and N. malicorticis was very low, as reported previously.     

Dynamics of pear-pathogenic Stemphylium vesicarium in necrotic plant residues in Dutch pear orchards

Brown spot disease on pear caused by Stemphylium vesicarium may affect leaves and fruits. Inoculum sources present on orchard floors play an important role in the epidemiology of pear brown spot. The pathogen can overwinter on plant residues and multiply and spread on the residues during the growing season. In the Netherlands, brown spot characteristically occurs only in a fraction of the orchards per season. Until now, no tools are available for Dutch pear growers to predict the risk of brown spot in specific orchards. As a consequence, preventive fungicide sprayings are common. The concentration of DNA of pear-pathogenic S. vesicarium was quantified by a specific TaqMan-PCR assay for various types of plant residues present on orchard floors to evaluate their importance as potential inoculum source. The pathogen was often found in residues of pear leaves, grasses and weeds, but only occasionally in mummies and prunings. Studies of the population dynamics showed that S. vesicarium decreased in dead pear leaves during early winter whereas pathogen populations developed with irregular pattern during the growing season on residues of weeds and grasses. Based on DNA concentrations of S. vesicarium in plant residue samples taken in 78 to 106 orchards in the springs of 2010, 2011 and 2012, the risk of brown spot development could be predicted for individual orchards. Such a risk prediction will allow growers to adapt their fungicide spray schedules to avoid unnecessary sprays in low-risk orchards.     

Pathogenicity of <Emphasis Type="Italic">Stemphylium vesicarium</Emphasis> from different hosts causing brown spot in pear

Stemphylium vesicarium (teleomorph: Pleospora herbarum) is the causal agent of brown spot disease in pear. The species is also able to cause disease in asparagus, onion and other crops. Saprophytic growth of the fungus on plant debris is common. The objective of this study was to investigate whether isolates of S. vesicarium from different hosts can be pathogenic to pear. More than hundred isolates of Stemphylium spp. were obtained from infected pear fruits, dead pear leaves, dead grass leaves present in pear orchard lawns as well as from necrotic leaf parts of asparagus and onion. Only isolates originating from pear orchards, including isolates from dead grass leaves, were pathogenic on pear leaves or fruits in bioassays. Non-pathogenic isolates were also present in pear orchards. Stemphylium vesicarium from asparagus or onion, with one exception, were not pathogenic to pear. Analysis of the genetic variation between isolates using Amplified Fragment Length Polymorphism (AFLP) showed significant concordance with host plants. Isolates from asparagus or onion belonged to clusters separate from the cluster with isolates from pear or grass leaves collected in pear orchards. Multilocus sequencing of a subset of isolates showed that such isolates were similar to S. vesicarium.     

The relative importance of conidia and ascospores as primary inoculum of Venturia inaequalis in a southeast England orchard

Apple scab, caused by Venturia inaequalis, can lead to large losses of marketable fruit if left uncontrolled. The disease appears in orchards during spring as lesions on leaves. These primary lesions are caused by spores released at bud burst from overwintering sources; these spores can be sexually produced ascospores from the leaf litter or asexual conidia from mycelium in wood scab or within buds. The relative importance of conidia and ascospores as primary inoculum were investigated in an orchard in southeast England, UK. Potted trees not previously exposed to apple scab were placed next to (c. 1 m) orchard trees to trap air‐dispersed ascospores. Number and position of scab lesions were assessed on the leaves of shoots from both the potted trees (infection by airborne ascospores) and neighbouring orchard trees (infection by both ascospores and splash‐dispersed, overwintered conidia). The distribution and population similarity of scab lesions were compared in the two tree types by molecular analysis and through modelling of scab incidence and count data. Molecular analysis was inconclusive. Statistical modelling of results suggested that conidia may have contributed approximately 20–50% of the primary inoculum in early spring within this orchard: incidence was estimated to be reduced by 20% on potted trees, and lesion number by 50%. These results indicate that, although conidia are still a minority contributor to primary inoculum, their contribution in this orchard is sufficient to require current management to be reviewed. This might also be true of other orchards with a similar climate.     

Necrotrophic behaviour of Erwinia amylovora in apple and tobacco leaf tissue

An important issue related to the epidemiology of fire blight, a devastating disease of apples and pears, is how its causal agent, the bacterium Erwinia amylovora, survives and disseminates in the environment. Almost no information is available on the possibility of this pathogen overwintering as a necrotroph. In this study, bacterial survival in dead apple and tobacco (a non‐host) leaf tissues was addressed. In necrotized leaves collected 5, 6, 7 and 8 months following shoot inoculation of apple trees, viable E. amylovora cells were present in over 50% of samples from the midrib and in over 10% of samples from lateral veins, but were never found in parenchyma. Using a PCR‐based method, pathogen DNA was detected in more than 50% of samples that were found to be free of viable cells by conventional plating out. However, PCR analysis was insufficient to distinguish between the DNA of viable and dead bacteria. Sugars appropriate for bacterial growth were found in dead apple leaves. In spot‐inoculated attached apple and tobacco leaves, a remarkable increase in the bacterial population was observed in lesions that developed as a hypersensitive response (HR). As in other necrotrophic interactions, bacterial proliferation was associated with massive hydrogen peroxide production and progression toward plant cell death. The results indicate that E. amylovora has an ability to survive as a semi‐necrotroph or necrotroph, which allows for overwintering in dead apple leaves.     

Effects of leaf position on reflectance,transmittance and absorption of red and far‐red light in tomato,Chenopodium album and Amaranthus retroflexus leaves

Leaf optical properties can play an important role in determining the red/far‐red light ratio, a signal of impending competition, in plant canopies. Knowledge of leaf optical properties and factors affecting them is important in understanding of the impacts of red/far‐red ratio in agroecosystems. Effects of leaf position on the plant stem on their optical properties at 660 and 730 nm were studied in tomato and two weeds Chenopodium album and Amaranthus retroflexus. Leaf position on stem strongly influenced leaf optical properties. Reflectance and transmittance were generally lower for the C. album and A. retroflexus leaves at higher positions on the stem, except for reflectance at 730 nm in C. album, which did not change. Reflectance was not affected in tomato. Transmittance generally decreased for leaves at higher positions. Red/far‐red ratios of reflected (R_ratio) and transmitted (T_ratio) light generally decreased in all species, except R_ratio in tomato, where it increased slightly at higher positions. These effects were greater in A. retroflexus compared with C. album and tomato. Changes in these ratios were partly explained by chlorophyll content and leaf mass per area. The results show that leaf position on plant stem influences leaf optical properties in tomato and two weeds and this effect differed between species. These influences and the differences among species could modify red/far‐red ratios in canopies comprising these species, which could influence their growth and inter‐plant interactions.     

Effects of selective cutting and timing of herbicide application on growth and development of Cirsium arvense in spring barley

Recently, a new implement for controlling weeds in cereals (CombCut) has been developed. It cuts weeds in growing cereals without damaging them by using the physical differences (in height, stem thickness, straw stiffness and branching pattern) between crops and weeds. To evaluate and compare the effects of selective cutting with different timings of herbicide application on Cirsium arvense in spring barley, a randomised block experiment was conducted in Sweden in 2015–2017, in a field with a naturally occurring C. arvense population. Treatments consisted of control (C), herbicide application at 4–5‐leaf stage of C. arvense (H1), herbicide application at 8–10‐leaf stage (H2) and selective cutting at 10‐leaf stage (S). The treatments were performed in 2015 and repeated in 2016 in the same plots, and a final evaluation was performed in 2017. Compared to the control, S, H1 and H2 were equally efficient in reducing above‐ground biomass production of C. arvense and increasing spring barley grain yield per unit area. The number of C. arvense shoots per area was, however, higher in S compared to H1 and H2. No differences in control effects on shoot number were observed between H1 and H2. Our study indicates that (i) selective cutting (S) reduces C. arvense equally efficient as herbicide application and (ii) early herbicide spraying is as efficient as spraying later in the season.     

Phenotypic,molecular and pathogenic characterization of Phlyctema vagabunda,causal agent of olive leprosy

Olive leprosy, caused by the fungus Phlyctema vagabunda, is a classic fruit rot disease widespread in the Mediterranean basin. From 2009 to 2013, new disease symptoms consisting of small circular necrotic leaf lesions, coin branch canker and shoot dieback were observed in Spanish and Portuguese olive orchards showing intense defoliation. Phlyctema‐like anamorphs were consistently isolated from leaves and shoots with symptoms. Representative isolates from affected leaves, shoots and fruits were characterized based on morphology of colonies and conidia, optimum growth temperature and comparison of DNA sequence data from four regions: ITS, tub2, MIT and rpb2. In addition, pathogenicity tests were performed on apple and olive fruits, and on branches and leaves of olive trees. Maximum mycelial growth rate ranged between 0.54 and 0.73 mm per day. Conidia produced on inoculated apple fruits showed slight differences in morphology among the representative fungal isolates evaluated. Phylogenetic analysis clustered all of the Phlyctema‐like isolates in the same clade, identifying them as Phlyctema vagabunda. On fruits, influence of wounding, ripening and cultivar resistance was studied, with cv. Blanqueta being the most susceptible cultivar. On branches, a mycelial‐plug inoculation method reproduced olive leprosy symptoms and caused shoot dieback. On leaves, Koch's postulates were fulfilled and the pathogen caused characteristic necrotic spots and plant defoliation. This is the first time that the pathogenicity of P. vagabunda in olive leaves has been demonstrated.     

Traditional and commercial apple and pear cultivars as sources of resistance to fireblight1

In Hungary, fireblight research programmes were initiated on pear in 1999 and on apple in 2000, with the aim of evaluating the susceptibility or resistance of commercial cultivars. Sources of resistance for future breeding were also sought among traditional apple cultivars collected from Ukraine and pear cultivars in the Hungarian gene bank (Szigetcsép). Experiments were done under secure conditions. Inocula were mixtures of characteristic Erwinia amylovora isolates from pear and apple in Hungary. Host responses (symptom development, disease severity and multiplication rate of bacterial cells in host tissues) were assessed on shoots, flowers and fruits. About 30 pear and 30 apple cultivars, and 35 apple hybrids, were tested and grouped into four categories for pear and three for apple. Of the pear cultivars tested, 50% were susceptible, 30% moderately susceptible and only 10% of low susceptibility. Different plant organs occasionally displayed different responses. Members of the last two groups might serve as useful candidates for growing under IPM conditions. Among the traditional Hungarian varieties tested, we found high resistance in ‘Sikulai’ and ‘Szemes alma’, which could be used as sources of fireblight resistance in breeding programmes and also grown in organic orchards. Furthermore, among the offspring of the apple ‘Prima’ (scab‐resistant), we have found highly resistant lines.     

Integrated pest-management approaches in orchard,cereal and potato protection in Poland1

With its total area of 312,683 km² and a population of about 39 million people, the area of agricultural land per person in Poland is about 0.47 ha. The yields of basic crops are rather low and the losses due to pathogens, animal pests and weeds are high. The use of pesticides is about 1.0–1.3 kg a.i. per ha and this gives a good starting point for integrated pest management (IPM) in the production system of various crops. IPM in glasshouse crops reached the level of 500 ha per year. An IPM approach against codling moth (Cydia pomonella) in orchards allowed a reduction by 70% in the number of insecticide treatments. In the case of apple scab (Venturia haequalis), the IPM approach allowed a reduction in the number of fungicide treatments by 30% on an area of 100,000 ha. In respect to small grains, the IPM approach concerns rational use of fungicides and herbicides in winter wheat and barley. In potato protection, the IPM approach mainly concerns Colorado beetle (Leptinotarsa decemlineata), which at present is controlled by chemical insecticides on 77% of the potato area. Special research and demonstration IPM programmes sponsored by USDA/AID in Central and Eastern Europe will be operational during 1992/1995 and will concern orchards (apple, pear), small grains (wheat, barley) and potatoes.     

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.     

Effect of six sanitation treatments on leaf litter density, ascospore production of Venturia inaequalis and scab incidence in integrated and organic apple orchards

A two-year study was conducted to determine the effect of six sanitation treatments on leaf litter density (LLD), relative ascospore production of Venturia inaequalis and scab incidence on spur-leaf clusters, leaves and harvested fruits, on two cultivars with low and high scab susceptibilities, in Hungarian integrated and organic apple orchards. The following sanitation treatments were used: sprays of lime sulphur in autumn, collecting fallen leaves in autumn, straw mulch cover in late winter, sprays of lime sulphur followed by mulch cover, collecting fallen leaves followed by mulch cover, collecting fallen leaves followed by covering the orchard floor with plastic foil, and non-sanitized control. LLD decreased continuously in all treatment plots by 0–23% by mid-May in both orchards and years; however, LLD reduction was 1.4–4.2 times higher in the organic orchard compared to the integrated one. All treatments, except for the lime sulphur treatment, resulted in significant (P < 0.05) reduction of LLD and ascospore production in both the integrated and organic apple orchards compared to non-sanitized plots. The most efficient treatment was leaf collection combined with plastic foil cover, followed by leaf collection combined with mulch cover, leaf collection alone, mulch cover alone, and lime sulphur spray combined with mulch cover, with a reduction in the ascospore production of >95, 72–92, 56–79, 24–38, and 27–46%, respectively, in the mean of both orchards and years. However, only treatments of leaf collection applied alone, or in combination with mulch or with plastic foil cover reduced significantly (P < 0.05) leaf and/or fruit scab incidence by 18–57% compared to non-sanitized plots. These three leaf collection treatments are recommended in both integrated and organic orchards and the possibilities of successfully incorporating these methods into orchard management practices are interpreted.     

Transmission of ‘Candidatus Phytoplasma pyri’ by naturally infected Cacopsylla pyri to peach,an approach to the epidemiology of peach yellow leaf roll (PYLR) in Spain

Peach orchards in the northeast of Spain were severely affected in 2012 by a previously unreported disease in this area. The symptoms included early reddening, leaf curling, decline, abnormal fruits, and in some cases death of the peach trees. All the infected peach samples were positive for ‘Candidatus Phytoplasma pyri’, but none were infected by the ‘Ca. Phytoplasma prunorum’. In this work, potential vectors able to transmit ‘Ca. Phytoplasma pyri’ from pear to peach and between peach trees were studied and their infective potential was analysed at different times of the year. Transmission trials of the phytoplasma with potential vectors to an artificial feeding medium for insects and to healthy peach trees were conducted. Additionally, isolated phytoplasmas were genetically characterized to determine which isolates were able to infect peach trees. Results showed that the only insect species captured inside peach plots that was a carrier of the ‘Ca. Phytoplasma pyri’ phytoplasma was Cacopsylla pyri. Other insect species captured and known to be phytoplasma transmitters were present in very low numbers, and were not infected with ‘Ca. Phytoplasma pyri’ phytoplasma. A total of 1928 individuals of C. pyri were captured in the peach orchards, of which around 49% were phytoplasma carriers. All the peach trees exposed to C. pyri in 2014, and 65% in 2015, were infected by ‘Ca. Phytoplasma pyri’ 1 year after exposure, showing that this species is able to transmit the phytoplasma to peach. Molecular characterization showed that some genotypes are preferentially determined in peach.     

Genetic population structure and fungicide resistance of Botrytis cinerea in pear orchards in the Western Cape of South Africa

Botrytis cinerea isolates from pear blossoms (Pyrus communis) in South Africa were collected from four orchards in two production areas in the Western Cape. The cryptic species status based on vegetative‐incompatibility alleles of the Bc‐hch gene indicated that all the isolates belonged to B. cinerea. A microsatellite analysis of B. cinerea populations was performed to assess the genetic population structure. Total gene diversity (H) was high, with a mean of 0.69 across all populations. Some genotype flow was evident between orchards as indicated by the spread of microsatellite multilocus genotypes, in agreement with the moderate, but significant population differentiation among orchards (mean φ_PT = 0.118, P = 0.001). Index of association analyses (I_A and r?_d) suggest that the populations reproduce mostly asexually, even though mating type distribution did not differ significantly from a 1:1 ratio, suggesting frequency‐dependent selection. Isolates resistant to benomyl were evident in one orchard only. This orchard was also significantly differentiated from all other populations, suggesting infrequent localized selection for benomyl resistance. Overall, the findings of this study highlight the dangers of a mixed reproduction system, and stress the importance of regularly monitoring fungicide resistance levels towards developing more efficient management practices.     

Population structure of Valsa ceratosperma, causal fungus of Valsa canker, in apple and pear orchards

On the basis of mycelial compatibility, the population structure of Valsa ceratosperma, the causal fungus of Valsa canker on broadleaf trees, was investigated in apple and pear orchards in Japan. Field strains of V. ceratosperma from a single canker on trunks or scaffold limbs belonged to different mycelial compatibility groups. Thus, the population structure of this fungus was complex in most orchards. Because mycelia of strains originating from different conidia from the same pycnidium were compatible, infection by this fungus is thought to be ascospores.     

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.     

Characterization of Neofabraea vagabunda isolates causing apple bull's eye rot in Italy (Emilia‐Romagna region)

Neofabraea vagabunda is the prevalent cause of bull's eye rot, one of the main postharvest diseases of apple, in many producing areas, but its biology has not been studied in detail. The molecular identification, by DNA sequencing of the β‐tubulin region, of 41 isolates collected from apples showing bull's eye rot in the Emilia‐Romagna region confirmed N. vagabunda as the main species in Italy. A biological and morphological characterization of N. vagabunda isolates was performed in vitro. Assays at temperatures ranging from 0 to 30 °C carried out on 10 isolates demonstrated: (i) a marked influence of temperature on colony morphology, conidial production, conidial size and mycelial growth, showing the cold‐tolerant character of N. vagabunda; and (ii) that culture at 15 °C on tomato agar (TA) for 14 days is a rapid and reliable method to favour pathogen conidial production. Trials performed on 38 isolates using these incubation conditions recorded the presence of two N. vagabunda morphotypes, differing for colony morphology, conidial size, conidiomata formation and temperature requirement. The alkalizing ability of the pathogen during growth on TA was also demonstrated for the first time. The pathogenicity of 25 N. vagabunda isolates was proved in vivo on artificially infected Cripps Pink apples. A pH increase was also recorded in apple tissue infected by N. vagabunda isolates (on average 0.2 and 0.3 units of pH after 60 and 120 days of incubation, respectively), suggesting that the N. vagabunda transition from quiescence to necrotrophic colonization in apples could involve the secretion of alkalizing compounds.     

Ecophysiological characteristics of five weeds and a wheat crop in the Indo‐Gangetic Plains,India

The objective of this research was to compare selected ecophysiological parameters for a wheat crop found in the Indo‐Gangetic Plains of India and its five dominant weeds. The dominant and regionally ubiquitous weeds in the wheat field that was selected for the study were Anagallis arvensis, Chenopodium album, Melilotus albus, Phalaris minor and Rumex dentatus. Taller weeds, such as C. album and P. minor, constituted one group along with the crop, with a low photosynthetic rate, specific leaf area, leaf nitrogen mass basis, chlorophyll content, photosynthetic nitrogen‐use efficiency and leaf area ratio, in comparison to shorter weeds, such as A. arvensis, M. albus and R. dentatus, which formed another group with a high photosynthetic rate, specific leaf area, leaf nitrogen mass basis, chlorophyll content, photosynthetic nitrogen‐use efficiency and leaf area ratio. Interspecific variations in the photosynthetic rate were driven mainly by variability in the specific leaf area and leaf nitrogen content. The taller weeds and the crop had a low specific leaf area later in the season, whereas the smaller weeds had a relatively high specific leaf area, which might be an adaptation to the shaded environment below the canopy. The result indicates that any weed management in the wheat fields of the Indo‐Gangetic Plains will need two different approaches because of the different strategies followed by the two weed groups that were identified in the present study.     

Characterization of Botryosphaeriaceae species associated with diseased loquat (Eriobotrya japonica) in Spain

Loquat (Eriobotrya japonica) is an important subtropical fruit crop in Spain and other Mediterranean countries. In recent years, characteristic symptoms of branch canker and dieback have been observed in the main cultivated areas of loquat in Spain. The goal of this study was to identify and characterize the species of Botryosphaeriaceae associated with these symptoms. For this, 36 affected orchards were surveyed between 2010 and 2011 in six provinces of southeastern Spain. Eighty‐four isolates belonging to the family Botryosphaeriaceae were recovered from samples with symptoms. These isolates were characterized by means of phenotypical studies, DNA sequence analyses of the internal transcribed spacer (ITS) and part of the translation elongation factor 1‐α regions, and pathogenicity tests. Ten fungal species were identified: Diplodia malorum, Diplodia olivarum, Diplodia seriata, Diplodia pseudoseriata/Diplodia alatafructa, Diplodia sp., Dothiorella sarmentorum, Neofusicoccum mediterraneum, N. parvum, Spencermartinsia plurivora and S. viticola. In addition, Diplodia eriobotryicola and Dothiorella eriobotryae are newly described. The most frequent species isolated from cankers was D. seriata and, as far as is known, this is the first report of D. malorum, and species belonging to the complex D. pseudoseriata/D. alatafructa, in Spain. All species were pathogenic to 1‐year‐old loquat plants cv. Algerie, with Diplodia sp. and S. viticola as the most virulent.