Important Apple Diseases Research Articles

Cell Wall Carbohydrate Dynamics during the Differentiation of Infection Structures by the Apple Scab Fungus, Venturia inaequalis.

Scab, caused by the biotrophic fungal pathogen Venturia inaequalis, is the most economically important disease of apples. During infection, V. inaequalis colonizes the subcuticular host environment, where it develops specialized infection structures called runner hyphae and stromata. These structures are thought to be involved in nutrient acquisition and effector (virulence factor) delivery, but also give rise to conidia that further the infection cycle. Despite their importance, very little is known about how these structures are differentiated. Likewise, nothing is known about how these structures are protected from host defenses or recognition by the host immune system. To better understand these processes, we first performed a glycosidic linkage analysis of sporulating tubular hyphae from V. inaequalis developed in culture. This analysis revealed that the V. inaequalis cell wall is mostly composed of glucans (44%) and mannans (37%), whereas chitin represents a much smaller proportion (4%). Next, we used transcriptomics and confocal laser scanning microscopy to provide insights into the cell wall carbohydrate composition of runner hyphae and stromata. These analyses revealed that, during subcuticular host colonization, genes of V. inaequalis putatively associated with the biosynthesis of immunogenic carbohydrates, such as chitin and β-1,6-glucan, are downregulated relative to growth in culture, while on the surface of runner hyphae and stromata, chitin is deacetylated to the less-immunogenic carbohydrate chitosan. These changes are anticipated to enable the subcuticular differentiation of runner hyphae and stromata by V. inaequalis, as well as to protect these structures from host defenses and recognition by the host immune system. IMPORTANCE Plant-pathogenic fungi are a major threat to food security. Among these are subcuticular pathogens, which often cause latent asymptomatic infections, making them difficult to control. A key feature of these pathogens is their ability to differentiate specialized subcuticular infection structures that, to date, remain largely understudied. This is typified by Venturia inaequalis, which causes scab, the most economically important disease of apples. In this study, we show that, during subcuticular host colonization, V. inaequalis downregulates genes associated with the biosynthesis of two immunogenic cell wall carbohydrates, chitin and β-1,6-glucan, and coats its subcuticular infection structures with a less-immunogenic carbohydrate, chitosan. These changes are anticipated to enable host colonization by V. inaequalis and provide a foundation for understanding subcuticular host colonization by other plant-pathogenic fungi. Such an understanding is important, as it may inform the development of novel control strategies against subcuticular plant-pathogenic fungi.

Reclassification of the Main Causal Agent of Glomerella Leaf Spot on Apple into Colletotrichum chrysophilum in Southern Brazil and Uruguay.

Glomerella leaf spot (GLS) is one of the most important diseases of apple, affecting a wide range of economically important cultivars, particularly Golden Delicious and its descendants. Caused mainly by species of the Colletotrichum gloeosporioides species complex (CGSC), C. fructicola has been described as the most prevalent and aggressive species associated with GLS and apple bitter rot (ABR) in Brazil and Uruguay. Recently, new CGSC species, closely related to C. fructicola, have been identified causing ABR. To verify the accuracy of species identification within the CGSC, we aimed to reevaluate the identity of representative GLS-causing isolates from Brazilian and Uruguayan populations, previously identified as C. fructicola. Multilocus phylogenetic analysis based on APN2, ApMAT, CAL, GAPDH, GS, ITS, and TUB2 allocated these isolates in a monophyletic clade with C. chrysophilum. This species was first described as the causal agent of anthracnose in banana fruits in Brazil, and recent reports indicate its association with ABR in the United States. This is the first report of C. chrysophilum causing GLS disease on apple worldwide.

Control of Apple Fruit Rot Caused by Alternaria porri and Alternaria mali by Using Hot Water Treatment and Some Inorganic Salts

Apple rot is one of the most important apple diseases worldwide. The disease causes significant losses in both the quantity and quality of apple fruits. In this study, the antifungal activity of hot water treatment and salts were investigated against apple fruit rots caused by Alternaria porri and Alternaria mali. Linear growth of tested fungi was inhibited at 5ºC, however, growth was increased by increasing storage temperature degree from 15ºC to 25ºC and decreased at 35ºC. On the other hand, hot water treatment at 55ºC significantly reduced the decay index and severity of infection. When salts such as potassium bicarbonate (KCO3), calcium chloride (CaCl2), sodium bicarbonate (NaHCO3) and ammonium bicarbonate (NH4CO3) was applied, a significant reduction in linear growth and fruit rot incidence was noticed using potassium bicarbonate. Calcium chloride, ammonium bicarbonate and sodium bicarbonate greatly inhibited growth of A. porri and A. mali. The most effective inhibitor of fruit decay was potassium bicarbonate and calcium chloride.

Podosphaera leucotricha (Ellis et Everhart) Salmon - prouzrokovač pepelnice jabuke

Apple powdery mildew, caused by Podosphaera leucotricha, is one of the economically most important apple diseases. It is present in all regions where the apple is grown, and it can cause production loss of up to 50%. The appearance of white mycelium on leaves, flowers and shoots is the typical symptom of this disease. If the disease is not managed properly, fruits are not formed, causing direct yield loss. Furthermore, fruit russet appears on the infected fruit, resulting in decreased market value. Disease intensity mostly depends on ecological factors and the susceptibility of the apple variety. Nowadays, significant efforts are being made in order to develop resistant apple varieties, but this goal is yet to be achieved. Management of apple powdery mildew in commercial production is based on chemical control measures, but due to health and environmental concerns, as well as the development of fungicide-resistant populations of the pathogen, it is necessary to find effective biological control methods as an alternative to chemicals.

Diseases of Apple (Malus domestica) and Their Management

Apple (Malus domestica) is the most important remunerative temperate fruit crop grown in the North Western Himalayan region of India. Apple orchards in India are under constant threat from a large number of fungal and viral pathogens. Appropriate and timely deployment of disease management depends on early disease detection. The most common diseases of apples are apple scab, powdery mildew, Alternaria leaf spot, Marsonina leaf spot, cankers, root rot, collar rot and mosaic. Depending on the incidence and severity of infection by diseases impacts range from unappealing cosmetic appearance low marketability, and poor quality of fruit, to decreased yield or complete loss of fruit or trees, causing huge economic losses. Early pest and disease detection are critical for appropriate and timely deployment of disease and pest management programs. Modern high-density apple orchards, which are usually made up of highly susceptible cultivars, are particularly vulnerable to the rapid spread of pathogens, potentially killing the entire orchard. Here we review some important diseases of apple and their management.

Investigating the Distribution of Strains of Erwinia amylovora and Streptomycin Resistance in Apple Orchards in New York Using Clustered Regularly Interspaced Short Palindromic Repeat Profiles: A 6-Year Follow-Up

Fire blight, caused by the bacterium Erwinia amylovora, is one of the most important diseases of apple. The antibiotic streptomycin is routinely used in the commercial apple industries of New York (NY) and New England to manage the disease. In 2002 and again, from 2011 to 2014, outbreaks of streptomycin resistance (SmR) were reported and investigated in NY. Motivated by new grower reports of control failures, we conducted a follow-up investigation of the distribution of SmR and E. amylovora strains for major apple production regions of NY over the last 6 years (2015 to 2020). Characterization of clustered regularly interspaced short palindromic repeat (CRISPR) profiles revealed that a few "cosmopolitan" strains were widely prevalent across regions, whereas many other "resident" strains were confined to one location. In addition, we uncovered novel CRISPR profile diversity in all investigated regions. SmR E. amylovora was detected only in a small area spanning two counties from 2017 to 2020 and was always associated with one CRISPR profile (41:23:38), which matched the profile of SmR E. amylovora, discovered in 2002. This suggests the original SmR E. amylovora was never fully eradicated and went undetected because of several seasons of low disease pressure in this region. Investigation of several representative isolates under controlled greenhouse conditions indicated significant differences in aggressiveness on 'Gala' apples. Potential implications of strain differences include the propensity of strains to become distributed across wide geographic regions and associated resistance management practices. Results from this work will directly influence sustainable fire blight management recommendations for commercial apple industries in NY state and other regions.

Optimizing the Integration of a Biopesticide (Bacillus subtilis QST 713) with a Single-Site Fungicide (Benzovindiflupyr) to Reduce Reliance on Synthetic Multisite Fungicides (Captan and Mancozeb) for Management of Apple Scab.

Apple scab is one of the most economically important diseases of apple in temperate production regions. In the absence of durable host resistance in commercially preferred cultivars, considerable applications of fungicides are needed to manage this disease. With the sequential development of resistance to nearly all classes of single-site fungicides in the apple scab pathogen Venturia inaequalis, synthetic multisite fungicides, such as mancozeb and captan, often comprise the core of chemical management programs for apple scab. Although these fungicides have demonstrable benefits for both disease and fungicide resistance management, the sustainability movement within agriculture aims to reduce reliance on such fungicides because of their broader environmental impacts. In this study, we establish a framework to enhance the feasibility of chemical management programs that do not rely on use of synthetic multisite protectant fungicides to manage apple scab. Specifically, we wish to evaluate chemical programs that integrate the biopesticide Bacillus subtilis QST 713 (Serenade Opti) in rotation with benzovindiflupyr (Aprovia), a single-site fungicide belonging to the class of succinate dehydrogenase inhibitors (SDHI), to circumvent the need for applications of synthetic multisite fungicides. During implementation of these programs, disease incidence data were taken at biweekly intervals. Regardless of the seasonal challenges presented in the 2 years of this study, when Bacillus subtilis QST 713 was used in place of captan and mancozeb mixtures, we did not observe any significant differences (P > 0.05) in development of apple scab symptoms between any of the management programs for the vertical axis or super spindle orchards in either year. This potential for substituting synthetic multisite fungicides with biopesticides is best realized when the programs are used with a decision support system in a super spindle planting system, where trees have reduced canopy densities. This 2-year study shows the potential to achieve adequate disease control using the integration of SDHI fungicides and biological controls without the use of synthetic multisite fungicides.

CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis

Apple scab, caused by the fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Using the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN) as a target for inactivation, which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference, we show, for the first time, that the CRISPR-Cas9 gene editing system can be successfully applied to the apple scab fungus. More specifically, using a CRISPR-Cas9 single guide RNA (sgRNA) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ∼16.7% gene inactivation efficiency. Notably, of the six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. In establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher gene function in this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.

Limited asymptomatic colonization of apple tree shoots by Neonectria ditissima following infection of leaf scars and pruning wounds

AbstractEuropean apple canker, caused by Neonectria ditissima, is an important disease of apple (Malus domestica). The fungus may reside in the tree without causing symptoms for up to a few years, thus making canker control difficult. Asymptomatic infections established in the nursery can result in severe canker outbreaks in newly established apple orchards. It has been suggested that N. ditissima might colonize the tree beyond the infection point during the asymptomatic stage. We investigated whether N. ditissima can colonize the internal tissues of apple shoots, both prior to and after visual symptoms. Apple trees were artificially inoculated via pruning wounds and leaf scars; then the pathogen was tracked at the inoculation point and beyond with isolation or real‐time quantitative PCR (qPCR). Before visual symptoms, N. ditissima could be detected in the infected pruning cut or leaf scar, but not at a distance of 10–15 mm from the entry point, or greater. Conversely, after symptom expression, the pathogen could be detected in the symptomless tissue at 10–15 mm from a canker lesion. This study demonstrated that the asymptomatic infection by N. ditissima can be detected using qPCR and that the pathogen does not grow systemically much beyond the initial entry point inside the plant before visual canker symptoms appear.

Pathogenic and biological characterisation of T-DNA insertional mutants of a Colletotrichum gloeosporioides casual organism of apple anthracnose

Anthracnose leaf spot caused by Colletotrichum gloeosporioides is an important disease of apples, resulting in serious damage to the fruit production. In this paper, the pathogenic and physiological characters of sixteen isolates and the wild isolate “Stj16” were studied. In the current study, we generated C. gloeosporioides strains expressing green fluorescence by introducing a GFP gene via an Agrobacterium tumefaciens-mediated transformation (ATMT). To confirm the subcellular localisation of the A2799 gene, an A2799gfp fusion expression mutant was constructed. After observation of the fusion expression, the A2799gfp fusion expression protein was located in the peroxisomes of the cell. The pathogenicity results showed that the mutants A4204, M44, A1919, A3638 and A1598 lost the pathogenic capability and virulence, however, the virulence of the mutants A1764, A439, A3885, G1183, A3144, A1649 and A2675 increased significantly to the apple fruits compared to the wild isolate “Stj16”. The biological study indicated that a Rose Bengal Agar (RBA) medium decreased the mycelium growth, but it can increase the sporulation for most of the isolates. The mutant A4204 does not grow well at pH 4.0 and pH 8.0, and mutant M44 just has the optimum growth at pH 8.0, and a 12 h light and 12 h dark condition stimulates the sporulation for most of the tested mutants, but the A1764 mutant more sporulated at regular dark conditions. All the mutants and “Stj16” grew vigorously at 25 °C–30 °C, for “Stj16”, it produced the highest number of conidia at 30 °C compared with the other temperatures. Based on the biological study, we found the best growing and sporulation conditions for all the tested isolates. The information generated in the present study will facilitate molecular research on this devastating fungus.

Functional characterization of the adenine transporter EaAdeP from the fire blight pathogen Erwinia amylovora and its effect on disease establishment in apples and pears.

Erwinia amylovora is the causal agent of fire blight, an economically important disease of apples and pears. As part of the infection process, Er. amylovora propagates on different plant tissues each with distinct nutrient environments. Here, the biochemical properties of the Er. amylovora adenine permease (EaAdeP) are investigated. Heterologous expression of EaAdeP in nucleobase transporter-deficient Escherichia coli strains, coupled with radiolabel uptake studies, revealed that EaAdeP is a high affinity adenine transporter with a Km of 0.43± 0.09 μM. Both Es. coli and Er. amylovora carrying extra copies of EaAdeP are sensitive to growth on the toxic analog 8-azaadenine. EaAdeP is expressed during immature pear fruit infection. Immature pear and apple fruit virulence assays reveal that an E. amylovora ΔadeP::Camr mutant is still able to cause disease symptoms, however, with growth at a lower level, indicating that external adenine is utilized in disease establishment.

A Genome Resource for the Apple Powdery Mildew Pathogen Podosphaera leucotricha.

Powdery mildew, caused by Podosphaera leucotricha, is an economically important disease of apple and pear trees. A single monoconidial strain (PuE-3) of this biotrophic fungus was used to extract DNA for Illumina sequencing. Data were assembled to form a draft genome of 43.8 Mb consisting of 8,921 contigs, 9,372 predicted genes, and 96.1% of complete benchmarking universal single copy orthologs (BUSCOs). This is the first reported genome sequence of P. leucotricha that will enable studies of the population biology, epidemiology, and fungicide resistance of this pathogen. Furthermore, this resource will be fundamental to uncover the genetic and molecular mechanisms of the apple-powdery mildew interaction, and support future pome fruit breeding efforts.

Colletotrichum acutatum complex isolated from apple flowers can cause bitter rot and Glomerella leaf spot

The Glomerella leaf spot (GLS) and bitter rot (BR) are important apple diseases in Brazil, caused by species of Colletotrichum, which are usually related with quiescent infection. This study aimed to detect quiescent infections of Colletotrichum spp. in flowers and in unripe fruits from apple commercial orchard cultivars Eva and Gala. The pathogen was observed in various structures of the flower in both cultivars. In unripe fruits, the pathogen was recovered only from ‘Eva’. Five isolates were obtained and were inoculated by mycelial (with and without wound) on ripe apple fruits and by conidia suspension (without wound) on ripe apple fruits and leaves of ‘Gala’. On inoculated fruits by mycelial, the isolates induced symptoms of BR, and on inoculated fruits by conidia without wound the isolates induced symptoms of GLS. On inoculated leaves, isolates induced symptoms of GLS. The isolates were characterized by cultural, morphological and pathogenic aspects. Detection of Colletotrichum spp. in asymptomatic apple flowers is epidemiologically important, since the flowers can be a route for infection in unripe fruits. This is the first investigation of quiescent infection of this pathogen on apple flowers.

First Report of Colletotrichum salicis Causing Bitter Rot of Apple in Italy.

Apple (Malus domestica Borkh.) is the most important fruit crop in South Tyrol (northern Italy), with a total production of 905.089 tons in 2019 (Chamber of Commerce, Industry, Crafts and Agriculture of Bozen-Bolzano 2020). Symptoms of bitter rot were observed on organic apples of the cultivar 'Roho 3615'/Evelina® collected in a packinghouse in South Tyrol at the end of March 2018 after approximately six months of storage in controlled atmosphere. Lesions were circular and light brown with orange conidial masses. Tissue samples were removed under aseptic conditions from surface-cleaned fruit at the margin between healthy and diseased pulp tissue, transferred to Petri dishes with potato dextrose agar (PDA), and incubated in the dark at room temperature for two weeks. Single spore cultures were obtained by adapting the procedure of Choi et al. (1999). Pure cultures were grown in quadruplicate on PDA at 20°C in the dark for two weeks. The colony appearance on the upper side was mostly flat with a distinct margin, the surface was covered with short, floccose aerial mycelium, and the color ranged from light gray to dark gray, while the reverse side appeared yellow. From each replicate culture, conidia were harvested, and the length and width of 50 randomly selected conidia were measured using a compound light microscope coupled to a digital camera (Leica DMLS, Leica Microsystems, Wetzlar, Germany). Conidia were cylindrical to fusiform, pointed at one end, and measured 10.0 to 19.5 × 2.5 to 5.0 μm (14.5 ± 1.9 × 3.9 ± 0.7 μm [mean ± SD]) in consistency with Damm et al. (2012). In order to determine the species of isolate 18-DSS-BS-EL-1-012, a multi-locus DNA sequence analysis was performed. Genomic DNA was extracted by following the protocol described by Cassago et al. (2002). Four loci, actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (HIS3) and the internal transcribed spacer (ITS) region of the rRNA operon were amplified by PCR and Sanger sequenced (Damm et al. 2012; Lévesque and de Cock 2004). The obtained DNA sequences of ACT, GAPDH, HIS3 and ITS were 186, 150, 317 and 505 bp long and were submitted to GenBank under the accession numbers MT347599, MT347600, MT347598 and MT337388, respectively. A MegaBLAST analysis resulted in 100% sequence identity at all four loci with a type culture of Colletotrichum salicis (CBS 607.94; GenBank accession numbers: JQ949781, JQ948791, JQ949451 and JQ948460), which belongs to the C. acutatum species complex (Damm et al. 2012). A pathogenicity test was performed with twelve 'Golden Delicious' apples by wounding the fruit with a sterile piercing tool and inoculating 20 μl of spore suspension (104 conidia per ml) from a 21-day-old PDA culture. Inoculated fruits were incubated for 21 days in a moist chamber at 20°C in the dark. The symptoms were recorded at 3, 5, 7, 10, 14 and 21 days post-inoculation (dpi). Symptoms appeared after 7 days on all inoculated fruits and resembled those observed on the original fruit, while mock-inoculated controls with sterile water remained symptomless. Fungal colonies resembling the original culture were re-isolated from lesions on the apple and plated on PDA. Their identity was confirmed by DNA sequence analysis of the ITS region, thereby proving Koch's postulates. Bitter rot occurs globally and is considered one of the most important diseases of apple that has the potential to cause significant crop losses (Sutton et al. 2014). Other Colletotrichum species have been commonly reported from apple in Europe, such as C. godetiae and C. fioriniae, whereas C. salicis has been reported solely in New Zealand and Belgium (Damm et al. 2012; Grammen et al. 2019). To the best of our knowledge, this is the first report of C. salicis causing bitter rot of apple in Italy.

Responses of Fuji (Malus domestica) and Shandingzi (Malus baccata) apples to Marssonina coronaria infection revealed by comparative transcriptome analysis

Marssonina leaf blotch of apple caused by Marssonina coronaria is one of the most important diseases of apple (Malus domestica) worldwide. Breeding and the appropriate utilization of disease-resistant varieties is the safest, most effective, most economical and environmentally friendly method for controlling the disease. Previous studies have demonstrated that Shandingzi (Malus baccata) is much more resistant to M. coronaria than Fuji (M. domestica). However, the molecular mechanisms of Fuji and Shandingzi resistance to M. coronaria infection remain unknown. In this study, the responses of Fuji and Shandingzi to M. coronaria infection were studied by comparative transcriptome analysis. As a result, 4898 and 2897 differentially expressed genes (DEGs) in Fuji and Shandingzi, respectively, were identified. Gene Ontology (GO) enrichment analysis of the co-expressed DEGs showed that 25 significant terms were enriched, and the unique DEGs in Shandingzi were mainly associated with the biotic stimulus response (GO: 0009607), defense response (GO: 0006952), stress response (GO: 0006950) and oxidoreductase activity (GO: 0016491). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs in both species of Rosaceae were enriched in plant-pathogen interactions, amino acid metabolism and other pathways. In the plant-pathogen interaction pathway, mitogen-activated protein kinase and Ca2+ signaling pathways were induced and enriched in both Shandingzi and Fuji when infected by M. coronaria. Furthermore, the percentages of R genes and disease resistance-related DEGs in Shandingzi were higher than those in Fuji. The qRT-PCR analysis results were consistent with the transcriptome results. The results provide a theoretical basis for breeding disease-resistant varieties.

Molecular diversity of "Candidatus Phytoplasma mali" strains associated with apple proliferation disease in Bulgarian germplasm collection.

A quarantine organism, "Candidatus Phytoplasma mali," is the causal agent of apple proliferation, one of the most important apple diseases in Europe. The genetic diversity of this pathogen in Central and Southern Europe has already been reported; however, almost no data exists from Eastern Europe. In this study, "Ca. P. mali" strains, which were identified in 14 apple trees from the Bulgarian germplasm collection, were characterized by restriction fragment length polymorphism (RFLP) and sequence analysis of four genomic loci. In total, nine distinct genetic lineages were recognized based on the combination of the following detected RFLP profiles: two profiles for the 16S-23S rDNA region (16SrX-A2, -A3), four profiles for the secY gene (one previously known: secY(X)-A, and three new: secY-C, secY-D, secY-E), three profiles for the rpl22-rps3 genes (rpX-A, rpX-B, rpX-F), and one profile for the nitroreductase- and rhodanese-like gene (AT-1). Phylogenetic analysis of the Bulgarian and other European "Ca. P. mali" strains based on 16S-23S rRNA gene sequences confirmed RFLP grouping, regardless of the phytoplasma origin. In a phylogenetic tree based on the secY data, only German strains formed separate clade from the other strains. The tree based on rp genes did not correspond to RFLP profiles. Unexpectedly, when using nitroreductase and rhodanese-like gene sequences, the Bulgarian strains clustered separately from the other European strains. Apart from the identification of different "Ca. P. mali" strains, the paper also recommends the unification of the rpX-subgroup nomenclature to avoid future confusions. Both aims of this paper provide valuable tools to understand the epidemiology of this quarantine pathogen.

Unraveling Colletotrichum species associated with Glomerella leaf spot of apple

Glomerella leaf spot (GLS), caused by Colletotrichum spp., is one of the most important diseases of apple. In this study we aimed to identify Colletotrichum species associated with GLS from different apple-producing areas in Southern Brazil and assess their differential pathogenicity towards apple. Sequencing and multilocus phylogenetic analysis using partial gene regions of ITS, GAPDH, TUB2, ACT and HIS3 allowed the identification of eighteen isolates of Colletotrichum fructicola, and only one of Colletotrichum nymphaeae. All isolates were pathogenic to apple leaves, while C. nymphaeae did not cause symptoms. Worldwide, our literature review showed nine Colletotrichum species reported associated with GLS in different countries: C. fructicola, C. nymphaeae and C. karstii in Brazil; C. fructicola and C. aenigma in China; C. fructicola, C. aenigma and C. siamense in Japan, C. fructicola, C. alienum, C. gloeosporioides, C. siamense, C. tropicale and C. fioriniae in the USA, and only C. fructicola in Uruguay. Our study confirms C. fructicola as the most frequent species causing GLS in Brazil and discusses the diversity and pathogenicity of GLS-causing species worldwide.

Effects of Temperature and Moisture on the Infection and Development of Apple Fruit Rot Caused by Phytophthora cactorum.

Phytophthora fruit rot, caused by Phytophthora cactorum, is an important disease of apple in China, often causing more than 50% fruit rot in rainy years. We examined the effects of temperature and moisture on the development of the disease and effects of the variables on zoospore release and germination, infection, and lesion development. In vitro, a temperature range of 5 to 20°C had no significant effects on zoospore release dynamics but did significantly affect the quantities of released zoospores. The largest quantity of zoospores was released at 9.9°C according to a fitted model. Zoosporangia released zoospores within 15 min at the test temperatures (0 to 20°C), which peaked at the fourth hour. Zoospores germinated in vitro, requiring free water, at temperatures from 5 to 35°C. The optimum germination temperature was 25.1°C according to a fitted model. The minimum wetness duration required for zoospores to complete the infection process and induce visible lesions on Fuji fruit was 0.40 h at the optimal temperature of 23.0°C according to the fitted model, whereas observed values were 4.5, 1.5, 0.5, 1.5 and 8.5 h at 10, 15, 20, 25, and 30°C, respectively. The number of zoospore infections on fruit at various temperatures and wetness durations were well fitted by the modified Weibull model; based on the model, the optimal temperature for zoospore infections was 23.0°C. Young apple fruit infected by zoospores developed visible lesions from 10 to 30°C, with a predicted optimum of 23.5°C; no lesions developed at 5 or 35°C. The shortest incubation period of the disease was 4 days. These results can be used to develop disease forecasting models for improved fungicide control.

An Erwinia amylovora yjeK mutant exhibits reduced virulence, increased chemical sensitivity and numerous environmentally dependent proteomic alterations.

The Gram-negative bacterium Erwinia amylovora causes fire blight, an economically important disease of apples and pears. Elongation factor P (EF-P) is a highly conserved protein that stimulates the formation of the first peptide bond of certain proteins and facilitates the translation of certain proteins, including those with polyproline motifs. YjeK and YjeA are two enzymes involved in the essential post-translational β-lysylation of EF-P at a conserved lysine residue, K34. EF-P, YjeA and YjeK have been shown to be essential for the full virulence of Escherichia coli, Salmonella species and Agrobacterium tumefaciens, with efp, yjeA and yjeK mutants having highly similar phenotypes. Here, we identified an E.amylovora yjeK::Tn5 transposon mutant with decreased virulence in apple fruit and trees. The yjeK::Tn5 mutant also showed pleiotropic phenotypes, including reduced growth in rich medium, lower extracellular polysaccharide production, reduced swimming motility and increased chemical sensitivity compared with the wild-type, whilst maintaining wild-type level growth in minimal medium. All yjeK::Tn5 mutant phenotypes were complemented intrans with a plasmid bearing a wild-type copy of yjeK. Comprehensive, quantitative proteomics analyses revealed numerous, environmentally dependent changes in the prevalence of a wide range of proteins, in higher abundance and lower abundance, in yjeK::Tn5 compared with the wild-type, and many of these alterations could be linked to yjeK::Tn5 mutant phenotypes. The environmental dependence of the yjeK::Tn5 mutant proteomic alterations suggests that YjeK could be required for aspects of the environmentally dependent regulation of protein translation. YjeK activity may be critical to overcoming stress, including the challenging host environment faced by invading pathogenic bacteria.

Seasonal susceptibility of apple trees to <i>Neonectria ditissima</i> wound infections

European canker, caused by Neonectria ditissima, is an important disease of apple in the temperate climate of southern Brazil. Monthly inoculations of this fungus were carried out on various wound types on cvs ‘Gala’ and ‘Fuji’ over two productive cycles to better understand the seasonal susceptibility of apple trees. Wound types were: bud scars (September), petal scars (October), fruit-thinning scars (November), leaf scars (from November to May), fruit-picking wound scars for ‘Gala’ (February) and ‘Fuji’ (March), and pruning wounds (June to August). One hundred plants of each cultivar were used and inoculation sites were monitored periodically for symptom expression. At each assessment, visible lesions were counted and pruned, and wounds were painted. Differences in wound susceptibility occurred throughout the year. Most lesions resulted from inoculations of pruning wounds. Inoculations performed in spring on bud and petal scars resulted in lower disease expression than inoculations later in the season.